Validation of the Thermo Scientific™ SureTect™ Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus PCR Assay for the Detection of 
Vibrio species cause an estimated 80 000 cases of vibriosis annually, of which approximately 100 are fatal in the US ( ). Vibriosis is contracted mainly through consumption of raw and improperly cooked seafood, with Vibrio cholerae , V. parahaemolyticus , and V. vulnificus the predominant causative species. Prevalence of these pathogenic Vibrio spp. has increased notably recently due to warming sea-surface temperature, which in turn has led to a global rise in cases of vibriosis ( ). Vibriosis is specifically concerning to high-risk individuals, such as those undergoing immunosuppressive treatment, due to limited available treatment options in conjunction with rapid onset of the disease, leading to a significantly higher mortality rate compared to healthy adults ( ). Lack of treatment options, rapid disease progression, increasing wide-range geographical distribution of Vibrio , and the annually increasing prevalence of immunocompromised adults in the US underscores the importance of preventative measures needed to detect these strains of concern and then implement corrective actions ( , ). Rapid detection of the main causative species in seafood is important in protecting public health and ensuring rapid throughput and release of products after processing that are pathogen-free. Culture media-based reference methods can take up to 24 h to return negative or presumptive positive results, and up to a further 48 h to confirm suspect colonies, whereas the Thermo Scientific SureTect Vibrio cholerae, V. parahaemolyticus, and V. vulnificus PCR Assay method is designed to detect the presumptive presence of V. cholerae , V. parahaemolyticus , and V. vulnificus in seafood samples in as little as 9–17 h. The sample screening result is available the next day, with flexibility offered during confirmation with multiple different selective agars. Thereafter, characteristic colonies can be biochemically confirmed. 

The SureTect Vibrio PCR Assay method is used in conjunction with either the Applied Biosystems™ 7500 Fast Real-Time Food Safety PCR Instrument with Applied Biosystems™ RapidFinder™ Express Software (version 2.0 or higher) or the Applied Biosystems™ QuantStudio™ 5 Real-Time Food Safety PCR instrument with Thermo Scientific™ RapidFinder Analysis Software (version 1.1 or higher) for the multiplex detection of V. cholerae, V. parahaemolyticus, or V. vulnificus in seafood samples ( ). The SureTect Vibrio PCR Assay is supplied as a kit containing all necessary reagents to conduct the sample lysis, including pre-filled lysis tubes and lyophilized PCR pellets as well as all necessary PCR reagents (target-specific primers, dye-labeled probes, and PCR master mix components) to easily conduct the PCR analysis. PCR probes are short oligonucleotides with a quencher molecule at one end that, when not bound to target DNA, greatly reduces fluorescence from the dye label at the opposite end of the probe molecule. The oligonucleotides target unique DNA sequences, including three unique targets for V. cholerae , V. parahaemolyticus , and V. vulnificus . If any of the strains are present, the target DNA sequences will be amplified and the increasing fluorescent signal generated will be detected by the 7500 Fast or the QuantStudio PCR Instrument and interpreted by the respective software. In addition to detecting any target DNA, the PCR pellets contain probes, primers, and DNA templates for an internal positive control (IPC). During PCR cycling, the IPC template is amplified regardless of the presence of any target DNA. The probe used for the IPC, labeled with a different-colored fluorescent dye than the probes used within the assay to detect the target DNA, can be detected by either the 7500 Fast or the QuantStudio 5 PCR Instrument through a separate dye channel. If there is no presence of target DNA, the presence of the IPC amplification curve indicates that the PCR process has occurred successfully. The PCR probes used in the SureTect Vibrio PCR Assay are based on TaqMan ® PCR technology. Results are achieved approximately 80 min after loading the prepared sample into either PCR instrument and are displayed via the appropriate instrumentational software on the attached computer screen as simple positive or negative symbols with an attached PCR amplification plot that is easily accessible for review. All results interpreted by the software can be reported, stored, printed, and downloaded as required by the user. 

Analyte(s).—Vibrio cholerae , Vibrio parahaemolyticus , and Vibrio vulnificus . Matrixes. —Up to 50 g raw tuna, raw mussels, green-lipped mussel extract, and salmon roll with cream cheese and up to 125 g cooked shrimp. Summary of validated performance claims .—The study data were unable to find a significant difference between the SureTect Vibrio PCR Assay and the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM), Chapter 9 (2004), Vibrio ( ) reference method for raw mussels, green-lipped mussel extract, and cooked shrimp. The SureTect method detected significantly more positive results for raw tuna and salmon roll with cream cheese than the BAM method. The study data were unable to find a significant difference between the SureTect Vibrio PCR Assay and the ISO 21872-1:2017 Microbiology of the food chain—Horizontal method for the determination of Vibrio spp. – Part 1: Detection of potentially enteropathogenic Vibrio parahaemolyticus, Vibrio cholerae, and Vibrio vulnificus ( ) reference method for raw tuna and raw mussels. 

Probability of detection (POD).— The proportion of positive analytical outcomes for a qualitative method for a given matrix at a given analyte level or concentration. POD is concentration dependent. Several POD measures can be calculated: POD R (reference method POD), POD C (confirmed candidate method POD), POD CP (candidate method presumptive result POD), and POD CC (candidate method confirmation result POD). Difference of probabilities of detection (dPOD).— Difference of probabilities of detection is the difference between any two POD values. If the confidence interval (CI) of a dPOD does not contain zero, then the difference is statistically significant at the 5% level. 

Test Kit Information Kit name .—Thermo Scientific SureTect Vibrio cholerae, V. parahaemolyticus, and V. vulnificus PCR Assay. Cat. No .—Catalog numbers A44253, A56837. Ordering information .— ( 1 ) USA: Remel, Inc., part of Thermo Fisher Scientific, Santa Fe Dr, Lenexa, KS, 66215. Tel: (800) 255-6730; ( 2 ) Europe: Oxoid Ltd, part of Thermo Fisher Scientific, Wade Rd, Basingstoke, Hampshire, RG24 8PW, UK. Tel: +44 1256 841144; ( 3 ) Asia/Pacific/China: Thermo Fisher Scientific, Thermo Fisher Biochemicals (Beijing) Ltd, 3rd Floor, 28 Yuhua Rd, Area B, Tianzhu Airport Industrial Zone, Beijing 101312, China; ( 4 ) Australia: Thermo Fisher Scientific Australia Pty Ltd, 20 Dalgleish St, Thebarton, Adelaide, South Australia, 5031. Test Kit Components Lysis Reagent 1 Tubes (clear colorless liquid containing fine white particles) .—Twelve strips of eight tubes. Thermo Scientific 96-Well Pierceable Seals .—Two sheets. Proteinase K (blue liquid) .—One tube. SureTect Vibrio cholerae, V. parahaemolyticus, and V. vulnificus PCR Tubes .—Twelve strips of eight tubes containing one pellet each. PCR Caps .—Twelve strips of eight caps. Additional Supplies and Reagents Thermo Scientific Oxoid™ Alkaline Saline Peptone Water (ISO) (Dehydrated) .—CM1117B or equivalent. Thermo Scientific Oxoid Thiosulfate-citrate-bile salts-sucrose (TCBS) Cholera Medium (Dehydrated) .—CM0333B or equivalent. CHROMagar™ Vibrio .—Available from Chromagar.com. Modified Cellobiose-Polymyxin B-Colistin (mCPC) agar .—Multiple laboratory suppliers. Cellobiose-Colistin (CC) Agar .—Multiple laboratory suppliers. Apparatus Unless otherwise indicated, all materials are available through Thermo Fisher Microbiology ( thermofisher.com ), MLS: Fisher Scientific ( fisherscientific.com ), or other major laboratory suppliers. Homogenizer laboratory blender or dilutor.— One of the following or equivalent: DB5000A, DB4100A, or DB4150A. Homogenizer bags appropriate for the sample type and size .—For DB4100A or DB4150A: DB4011A, DB4012A, DB4013A, DB4014A. Incubator fitted with racks for homogenizer bags, set to 35 ± 1°C . Disposable gloves . Variable volume single-channel pipet, 1 to 10 mL . 96-well rack .—For holding samples. Filtered pipet tips, 1 to 10 mL . Sample tubes, 1.5 mL . Single-channel pipet, 10 to 100 μL, or electronic adjustable spacing, multichannel pipet, 10 to 100 μL . Single-channel stepper pipet, 10 to 100 μL . Filtered pipet tips, 10 to 100 μL . Domed Lysis Tube Caps .—A56895. Thermo Scientific 96-Well Pierceable Seals .—A55331. MicroAmp™ Adhesive Film Applicator (for pierceable seals) .—4333183. Thermo Scientific PCR Strip Cutting Tool .—A55329. Thermo Scientific PCR Capping Tool .—A55327. Thermo Scientific PCR Uncapping Tool .—A55328. Applied Biosystems SimpliAmp™ Thermal Cycler .—A24811. SimpliAmp Strip Plate Tray .—A55333. Thermo Scientific 96-Well PCR Base .—A55330. QuantStudio 5 Food Safety Real-Time PCR Instrument, 0.1 mL block, with RapidFinder Analysis Software v.1.1 or later. For use with SureTect Vibrio cholerae, V. parahaemolyticus, and V. vulnificus PCR Assay and Pathogen Assay File: Vibrio Multiplex SureTect QS5 version 1.0 or later .—A36320 (desktop), A36328 (laptop). 7500 Fast Food Safety Real-Time PCR Instrument with RapidFinder Express Software v.2.0 or later . For use with SureTect Vibrio cholerae, V. parahaemolyticus, and V. vulnificus PCR Assay and Pathogen Assay File: V. cholerae Multiplex SureTect 1.0 or later.—A30304 (desktop), A30299 (laptop). QuantStudio 5 Strip Plate Tray .—A55332. 7500 Fast 96W Strip Plate Adaptor .—A46347. Armadillo™ Low-Profile PCR Strip Plate, 96-well, clear well .—AB2696. Ultra Clear qPCR Caps, strips of eight .—AB0866. Vortex mixer . Plate spinner . Eight-channel pipet, 10 to 100 μL . Filtered pipet tips, 10 to 100 μL . Safety Precautions General safety: Using the SureTect Vibrio PCR Assay in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device. Ensure that anyone using this product has received instructions in general safety practice for laboratories and the safety information provided in this document. Before handling chemicals, read and understand all applicable Safety Data Sheets (SDS) and use appropriate personal protective equipment (gloves, gowns, eye protection, etc.). Refer to the 7500 Fast Real-Time PCR System and QuantStudio 5 Real-Time qPCR System Instrument Manuals for guidelines on cleaning equipment and handling possible amplicon contamination. For disposal of uninoculated culture media or any reagents and materials included in the SureTect Vibrio cholerae, V. parahaemolyticus, and V. vulnificus PCR Assay and associated tests, refer to the manufacturer’s SDS and apply appropriate local guidelines. To minimize hazards, ensure that laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below. Consult the relevant SDS for specific precautions and instructions. Read and understand the SDS provided by the chemical manufacturer before storing, handling, or working with any chemicals or hazardous materials. To obtain SDS, see the “Documentation and Support” section in this document. Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (e.g., safety glasses, gloves, or protective clothing). Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with sufficient ventilation (e.g., fumehood). Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer cleanup procedures as recommended in the SDS. Handle chemical wastes in a fumehood. Ensure the use of primary and secondary waste containers. (A primary waste container holds the immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.) After emptying, seal the waste container with the cap provided. Characterize (by analysis if needed) the wastes generated from the particular application, reagents, and substrates used in your laboratory. Ensure that the waste is stored, transferred, transported, and disposed according to all local, state/provincial, and national regulations. Radioactive or biohazardous materials may require special handling, and disposal limitations may apply. Depending on the samples used on this instrument, the surface may be considered a biohazard.Use appropriate decontamination methods when working with biohazards. Conduct all work in properly equipped facilities with the appropriate safety equipment (e.g., physical containment devices). Safety equipment can also include items for personal protection, such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles. Individuals should be trained according to applicable regulatory and company/institution requirements before working with potentially biohazardous materials. Follow all applicable local, state/provincial, and national regulations. Vibrio spp. may be present in many food specimens. There are multiple routes of exposure to humans, with the primary hazards to laboratory personnel being direct contact, mucous membrane, ingestion, and percutaneous routes of infection from contaminated material. For potential contaminated materials, Containment Level 3 practices, containment equipment, and facilities are recommended for all activities. The following references provide general guidelines when handling biological samples in a laboratory environment: U.S. Department of Health and Human Services, Biosafety in Microbiological and Biomedical Laboratories (BMBL) , 6th Edition, HHS Publication No. (CDC) 300859, revised June 2020; found at: Biosafety in Microbiological and Biomedical Laboratories—6th Edition (cdc.gov) . World Health Organization, Laboratory Biosafety Manual, 3rd Edition, WHO/CDS/CSR/LYO/2004.11; found at: https://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf . General Preparation Prepare all media and use all confirmation kits according to the manufacturer’s instructions. Turn on the SimpliAmp Thermal Cycler and ensure that the required temperature cycle is set according to the manufacturer’s instructions. Allow Lysis Reagent and PCR tubes to reach ambient temperature (23 ± 5°C) before use by placing on the laboratory bench approximately 10 min before they are required. Sample Preparation For 125 g cooked shrimp, 1-in-10 ratio of sample to media. Add 1125 mL of single-strength Alkaline Saline Peptone Water (ASPW). Homogenize the sample for 30 s to 1 min, and incubate at 35.0 ± 1°C for 16–24 h. For 50 g raw tuna, 50 g raw mussels, and 50 g green-lipped mussel extract, 1-in-10 ratio of sample to media. Add 450 mL of single-strength ASPW. Homogenize the sample for 30 s to 1 min, and incubate at 35.0 ± 1°C for 16–24 h. For 50 g salmon roll with cream cheese, 1-in-10 ratio of sample to media. Add 450 mL of single-strength ASPW. Homogenize the sample for 30 s to 1 min, and incubate at 35.0 ± 1°C for 8–20 h. Analysis Lysis Equilibrate the Lysis Reagent 1 Tubes to room temperature (23.0 ± 5°C). Place the required number of Lysis Reagent 1 Tubes in a MicroAmp Splash-Free 96-Well Base and MicroAmp 96-Well Tray. Ensure that the liquid is collected at the bottom of each tube—flick the tubes in a downward motion or tap the tubes against the bench. Allow the tubes to remain at room temperature for approximately 10 min before opening. Remove the foil from each Lysis Reagent 1 Tube, and then add 10 μL of Proteinase K to the tube. Avoid contamination of the Proteinase K stock tube by using a new filtered pipet tip each time Proteinase K is withdrawn from the stock tube. Use a 10–100 μL repeat pipettor to reduce the number of tips required. Transfer 10 μL of the enriched sample to a lysis tube. For the negative extraction controls, transfer 10 μL of sterile enrichment media to a lysis tube. Ensure that the pipet tip reaches the bottom of the lysis tube to facilitate complete mixing of the sample with Lysis Reagent 1. Seal the tubes with the domed Lysis Tube Caps using the Thermo Scientific PCR Capping Tool or with the Thermo Scientific 96-Well Pierceable Seal using the MicroAmp Adhesive Film Applicator. To conduct the lysis procedure, incubate the samples in the Applied Biosystems SimpliAmp Thermal Cycler set to the parameters outlined in . Note: To prevent crushing of the tubes in the SimpliAmp Thermal Cycler, use the bottom piece from the MicroAmp 96-Well Tray/Retainer Set, or include at least four complete SureTect lysis tube strips. PCR Setup—Quantstudio 5 Instrument Use the RapidFinder Analysis Software to determine the Run Layout (plate layout) for the samples based on the information entered and create a run file. Refer to the online help in the software for more details. On the home page of the RapidFinder Analysis Software, select “Create Experiment,” and then “enter” or “edit” the well parameters. Select Vibrio Multiplex SureTect QS5 version 1.0 (or later) for the assay. Following the plate layout previously set up in the software, place the required number of SureTect Vibrio cholerae, V. parahaemolyticus, and V. vulnificus PCR Tubes (PCR tubes) in the MicroAmp 96-Well Tray for VeriFlex Block. Place the block on the MicroAmp Splash-Free 96-Well Base. Firmly press the PCR tubes to the tray, and then tap the tubes on the bench to ensure that the pellets are located at the bottom of the tubes. Allow the PCR tubes to remain on the bench for approximately 5 min, to bring to room temperature, and then open one strip of PCR tubes by removing the seal. PCR pellets are pale yellow. If the pellet has collapsed or is not pale yellow, do not use. If the pellet is not positioned at the bottom of a tube, gently move the pellet to the bottom of the tube with a sterile, empty pipet tip. Do not use a tip containing lysate. Uncap the lysis tubes using the decapping tool. Transfer 20 μL of the lysate or mock-purified sample (negative extraction control reaction) to the appropriate PCR tube to rehydrate the pellet. Tap the rack to ensure that the lysate is at the bottom of the tube and touching the pellet. Remove lysate from the top half of the liquid to ensure that no lysis particles are transferred from the lysis tube to the PCR tube as this can inhibit PCR from occurring—also be sure not to touch the pellet when adding the lysate. Seal the PCR tubes with the flat optical PCR caps provided with the kit. Ensure that the tubes are properly sealed by pressing down firmly over each opening. Do not use the capping tool to seal the PCR tubes. Mix the contents of all the PCR tubes thoroughly by vortexing for 10–15 s to ensure that the pellet is fully rehydrated. Spin the PCR tubes using a plate spinner for at least 10 s to remove bubbles and to collect the liquid at the bottom of the tube before placing in the PCR instrument. Start the run within 30 min of adding sample lysates to the PCR tubes. ( k )Open the instrument drawer. Use the MicroAmp 96-Well Tray for VeriFlex Block to transfer the tubes to the instrument in the same configuration as the plate layout in the software, and then close the instrument drawer. ( l )In the “Run” tab of the experiment file in RapidFinder Analysis Software, select the instrument’s serial number from the Instrument drop-down list. ( m )Click “Start Run,” and then follow the software prompts. PCR Setup—7500 Fast Instrument Use the RapidFinder Express Software to determine the Run Layout (plate layout) for samples based on the information entered and create a run file. Refer to the online help in the software for more details. On the main page of RapidFinder Express Software, select “Create/Edit a Run File,” and then “enter” or “edit” the Run File information at the prompts. Plate layouts can be manually customized in the software. Select “V. cholerae Multiplex SureTect 1.0 or later” for the assay. Following the plate layout previously set up in the software, place the required number of SureTect Vibrio cholerae, V. parahaemolyticus, and V. vulnificus PCR Tubes (PCR tubes) in a suitable rack with a PCR carry plate, and then tap the rack of tubes on the bench to ensure that the pellets are located at the bottom of the tubes. If required by the plate layout, place empty SureTect PCR tubes in the rack to balance the tray when the tubes are placed in the instrument. Allow the PCR tubes to remain on the bench for approximately 5 min, to bring to room temperature, and then open one strip of PCR tubes by removing the seal. PCR pellets are pale yellow. If the pellet has collapsed or is not pale yellow, do not use. If the pellet is not positioned at the bottom of a tube, gently move the pellet to the bottom of the tube with a sterile, empty pipet tip. Do not use a tip containing lysate. Uncap the lysis tubes using the decapping tool. Transfer 20 μL of the lysate or mock-purified sample (negative extraction control reaction) to the appropriate PCR tube to rehydrate the pellet. Tap the rack to ensure that the lysate is at the bottom of the tube and touching the pellet. Remove lysate from the top half of the liquid to ensure that no lysis particles are transferred from the lysis tube to the PCR tube as this can inhibit PCR from occurring. Do not touch the pellet when adding the lysate. Seal the PCR tubes with the flat optical PCR caps provided with the kit. Ensure that the tubes are properly sealed by pressing down firmly over each opening. Do not use the capping tool to seal the PCR tubes. Mix the contents of all PCR tubes thoroughly by vortexing for 10–15 s to ensure that the pellet is fully rehydrated. Spin the PCR tubes using a plate spinner for at least 10 s to remove bubbles and to collect the liquid at the bottom of the tube before placing in the PCR instrument. Start the PCR run within 30 min of adding sample lysates to the PCR tubes. ( j )In the RapidFinder Express Software, select Start Instrument Run on the main page, select the appropriate run file, and follow the software prompts. ( k )Use the PCR carry plate to transfer the tubes to the instrument in the same configuration as the run layout. Use the Precision Plate Holder for SureTect assays. Load empty SureTect PCR tube strips as directed by the software. ( l )Close the tray to the instrument and follow the RapidFinder Express Software prompts to start the run. Calculations, Interpretation, and Test Results Report For the QuantStudio 5 PCR Instrument: Data analysis is automated by the software. For detailed instructions and options for reporting, exporting, and storing results, see the Help function in the software. In the home screen of the RapidFinder Analysis Software, click Results, and then click the sub-tab for the desired view of the data. Summary—plate format Results—table format Details—amplification plot For the 7500 Fast PCR Instrument: Data analysis is automated by the software. In the RapidFinder Express Software, select View Results on the main page, select the appropriate run file, and follow the prompts to view results. To display a list of results in table format, click Table View. Select a sample, and then click View Details to see replicate information about samples. Confirmation Samples with positive PCR results must be confirmed by selective plating and further confirmed by using conventional tests described in the methods standardized by CEN or ISO (EN ISO 21872–1:2017) or BAM Chapter 9 from colonies (including the purification step). The confirmation step must start from the primary enrichment broth. To confirm presumptive positives using the alternative candidate confirmation method: Streak 10 μL of the enriched sample onto plates of TCBS agar and/or CHROMagar Vibrio, Modified mCPC agar, or CC agar. Incubate the plates at 34–38°C for 21–27 h under aerobic conditions. Pick and purify up to five isolated colonies using plates of nonselective saline nutrient agar. Biochemically confirm the colonies using test kits or one of the reference methods. 

Kit name .—Thermo Scientific SureTect Vibrio cholerae, V. parahaemolyticus, and V. vulnificus PCR Assay. Cat. No .—Catalog numbers A44253, A56837. Ordering information .— ( 1 ) USA: Remel, Inc., part of Thermo Fisher Scientific, Santa Fe Dr, Lenexa, KS, 66215. Tel: (800) 255-6730; ( 2 ) Europe: Oxoid Ltd, part of Thermo Fisher Scientific, Wade Rd, Basingstoke, Hampshire, RG24 8PW, UK. Tel: +44 1256 841144; ( 3 ) Asia/Pacific/China: Thermo Fisher Scientific, Thermo Fisher Biochemicals (Beijing) Ltd, 3rd Floor, 28 Yuhua Rd, Area B, Tianzhu Airport Industrial Zone, Beijing 101312, China; ( 4 ) Australia: Thermo Fisher Scientific Australia Pty Ltd, 20 Dalgleish St, Thebarton, Adelaide, South Australia, 5031. 

Lysis Reagent 1 Tubes (clear colorless liquid containing fine white particles) .—Twelve strips of eight tubes. Thermo Scientific 96-Well Pierceable Seals .—Two sheets. Proteinase K (blue liquid) .—One tube. SureTect Vibrio cholerae, V. parahaemolyticus, and V. vulnificus PCR Tubes .—Twelve strips of eight tubes containing one pellet each. PCR Caps .—Twelve strips of eight caps. 

Thermo Scientific Oxoid™ Alkaline Saline Peptone Water (ISO) (Dehydrated) .—CM1117B or equivalent. Thermo Scientific Oxoid Thiosulfate-citrate-bile salts-sucrose (TCBS) Cholera Medium (Dehydrated) .—CM0333B or equivalent. CHROMagar™ Vibrio .—Available from Chromagar.com. Modified Cellobiose-Polymyxin B-Colistin (mCPC) agar .—Multiple laboratory suppliers. Cellobiose-Colistin (CC) Agar .—Multiple laboratory suppliers. 

Unless otherwise indicated, all materials are available through Thermo Fisher Microbiology ( thermofisher.com ), MLS: Fisher Scientific ( fisherscientific.com ), or other major laboratory suppliers. Homogenizer laboratory blender or dilutor.— One of the following or equivalent: DB5000A, DB4100A, or DB4150A. Homogenizer bags appropriate for the sample type and size .—For DB4100A or DB4150A: DB4011A, DB4012A, DB4013A, DB4014A. Incubator fitted with racks for homogenizer bags, set to 35 ± 1°C . Disposable gloves . Variable volume single-channel pipet, 1 to 10 mL . 96-well rack .—For holding samples. Filtered pipet tips, 1 to 10 mL . Sample tubes, 1.5 mL . Single-channel pipet, 10 to 100 μL, or electronic adjustable spacing, multichannel pipet, 10 to 100 μL . Single-channel stepper pipet, 10 to 100 μL . Filtered pipet tips, 10 to 100 μL . Domed Lysis Tube Caps .—A56895. Thermo Scientific 96-Well Pierceable Seals .—A55331. MicroAmp™ Adhesive Film Applicator (for pierceable seals) .—4333183. Thermo Scientific PCR Strip Cutting Tool .—A55329. Thermo Scientific PCR Capping Tool .—A55327. Thermo Scientific PCR Uncapping Tool .—A55328. Applied Biosystems SimpliAmp™ Thermal Cycler .—A24811. SimpliAmp Strip Plate Tray .—A55333. Thermo Scientific 96-Well PCR Base .—A55330. QuantStudio 5 Food Safety Real-Time PCR Instrument, 0.1 mL block, with RapidFinder Analysis Software v.1.1 or later. For use with SureTect Vibrio cholerae, V. parahaemolyticus, and V. vulnificus PCR Assay and Pathogen Assay File: Vibrio Multiplex SureTect QS5 version 1.0 or later .—A36320 (desktop), A36328 (laptop). 7500 Fast Food Safety Real-Time PCR Instrument with RapidFinder Express Software v.2.0 or later . For use with SureTect Vibrio cholerae, V. parahaemolyticus, and V. vulnificus PCR Assay and Pathogen Assay File: V. cholerae Multiplex SureTect 1.0 or later.—A30304 (desktop), A30299 (laptop). QuantStudio 5 Strip Plate Tray .—A55332. 7500 Fast 96W Strip Plate Adaptor .—A46347. Armadillo™ Low-Profile PCR Strip Plate, 96-well, clear well .—AB2696. Ultra Clear qPCR Caps, strips of eight .—AB0866. Vortex mixer . Plate spinner . Eight-channel pipet, 10 to 100 μL . Filtered pipet tips, 10 to 100 μL . 

General safety: Using the SureTect Vibrio PCR Assay in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device. Ensure that anyone using this product has received instructions in general safety practice for laboratories and the safety information provided in this document. Before handling chemicals, read and understand all applicable Safety Data Sheets (SDS) and use appropriate personal protective equipment (gloves, gowns, eye protection, etc.). Refer to the 7500 Fast Real-Time PCR System and QuantStudio 5 Real-Time qPCR System Instrument Manuals for guidelines on cleaning equipment and handling possible amplicon contamination. For disposal of uninoculated culture media or any reagents and materials included in the SureTect Vibrio cholerae, V. parahaemolyticus, and V. vulnificus PCR Assay and associated tests, refer to the manufacturer’s SDS and apply appropriate local guidelines. To minimize hazards, ensure that laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below. Consult the relevant SDS for specific precautions and instructions. Read and understand the SDS provided by the chemical manufacturer before storing, handling, or working with any chemicals or hazardous materials. To obtain SDS, see the “Documentation and Support” section in this document. Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (e.g., safety glasses, gloves, or protective clothing). Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with sufficient ventilation (e.g., fumehood). Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer cleanup procedures as recommended in the SDS. Handle chemical wastes in a fumehood. Ensure the use of primary and secondary waste containers. (A primary waste container holds the immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.) After emptying, seal the waste container with the cap provided. Characterize (by analysis if needed) the wastes generated from the particular application, reagents, and substrates used in your laboratory. Ensure that the waste is stored, transferred, transported, and disposed according to all local, state/provincial, and national regulations. Radioactive or biohazardous materials may require special handling, and disposal limitations may apply. Depending on the samples used on this instrument, the surface may be considered a biohazard.Use appropriate decontamination methods when working with biohazards. Conduct all work in properly equipped facilities with the appropriate safety equipment (e.g., physical containment devices). Safety equipment can also include items for personal protection, such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles. Individuals should be trained according to applicable regulatory and company/institution requirements before working with potentially biohazardous materials. Follow all applicable local, state/provincial, and national regulations. Vibrio spp. may be present in many food specimens. There are multiple routes of exposure to humans, with the primary hazards to laboratory personnel being direct contact, mucous membrane, ingestion, and percutaneous routes of infection from contaminated material. For potential contaminated materials, Containment Level 3 practices, containment equipment, and facilities are recommended for all activities. The following references provide general guidelines when handling biological samples in a laboratory environment: U.S. Department of Health and Human Services, Biosafety in Microbiological and Biomedical Laboratories (BMBL) , 6th Edition, HHS Publication No. (CDC) 300859, revised June 2020; found at: Biosafety in Microbiological and Biomedical Laboratories—6th Edition (cdc.gov) . World Health Organization, Laboratory Biosafety Manual, 3rd Edition, WHO/CDS/CSR/LYO/2004.11; found at: https://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf . 

Prepare all media and use all confirmation kits according to the manufacturer’s instructions. Turn on the SimpliAmp Thermal Cycler and ensure that the required temperature cycle is set according to the manufacturer’s instructions. Allow Lysis Reagent and PCR tubes to reach ambient temperature (23 ± 5°C) before use by placing on the laboratory bench approximately 10 min before they are required. 

For 125 g cooked shrimp, 1-in-10 ratio of sample to media. Add 1125 mL of single-strength Alkaline Saline Peptone Water (ASPW). Homogenize the sample for 30 s to 1 min, and incubate at 35.0 ± 1°C for 16–24 h. For 50 g raw tuna, 50 g raw mussels, and 50 g green-lipped mussel extract, 1-in-10 ratio of sample to media. Add 450 mL of single-strength ASPW. Homogenize the sample for 30 s to 1 min, and incubate at 35.0 ± 1°C for 16–24 h. For 50 g salmon roll with cream cheese, 1-in-10 ratio of sample to media. Add 450 mL of single-strength ASPW. Homogenize the sample for 30 s to 1 min, and incubate at 35.0 ± 1°C for 8–20 h. 

Lysis Equilibrate the Lysis Reagent 1 Tubes to room temperature (23.0 ± 5°C). Place the required number of Lysis Reagent 1 Tubes in a MicroAmp Splash-Free 96-Well Base and MicroAmp 96-Well Tray. Ensure that the liquid is collected at the bottom of each tube—flick the tubes in a downward motion or tap the tubes against the bench. Allow the tubes to remain at room temperature for approximately 10 min before opening. Remove the foil from each Lysis Reagent 1 Tube, and then add 10 μL of Proteinase K to the tube. Avoid contamination of the Proteinase K stock tube by using a new filtered pipet tip each time Proteinase K is withdrawn from the stock tube. Use a 10–100 μL repeat pipettor to reduce the number of tips required. Transfer 10 μL of the enriched sample to a lysis tube. For the negative extraction controls, transfer 10 μL of sterile enrichment media to a lysis tube. Ensure that the pipet tip reaches the bottom of the lysis tube to facilitate complete mixing of the sample with Lysis Reagent 1. Seal the tubes with the domed Lysis Tube Caps using the Thermo Scientific PCR Capping Tool or with the Thermo Scientific 96-Well Pierceable Seal using the MicroAmp Adhesive Film Applicator. To conduct the lysis procedure, incubate the samples in the Applied Biosystems SimpliAmp Thermal Cycler set to the parameters outlined in . Note: To prevent crushing of the tubes in the SimpliAmp Thermal Cycler, use the bottom piece from the MicroAmp 96-Well Tray/Retainer Set, or include at least four complete SureTect lysis tube strips. PCR Setup—Quantstudio 5 Instrument Use the RapidFinder Analysis Software to determine the Run Layout (plate layout) for the samples based on the information entered and create a run file. Refer to the online help in the software for more details. On the home page of the RapidFinder Analysis Software, select “Create Experiment,” and then “enter” or “edit” the well parameters. Select Vibrio Multiplex SureTect QS5 version 1.0 (or later) for the assay. Following the plate layout previously set up in the software, place the required number of SureTect Vibrio cholerae, V. parahaemolyticus, and V. vulnificus PCR Tubes (PCR tubes) in the MicroAmp 96-Well Tray for VeriFlex Block. Place the block on the MicroAmp Splash-Free 96-Well Base. Firmly press the PCR tubes to the tray, and then tap the tubes on the bench to ensure that the pellets are located at the bottom of the tubes. Allow the PCR tubes to remain on the bench for approximately 5 min, to bring to room temperature, and then open one strip of PCR tubes by removing the seal. PCR pellets are pale yellow. If the pellet has collapsed or is not pale yellow, do not use. If the pellet is not positioned at the bottom of a tube, gently move the pellet to the bottom of the tube with a sterile, empty pipet tip. Do not use a tip containing lysate. Uncap the lysis tubes using the decapping tool. Transfer 20 μL of the lysate or mock-purified sample (negative extraction control reaction) to the appropriate PCR tube to rehydrate the pellet. Tap the rack to ensure that the lysate is at the bottom of the tube and touching the pellet. Remove lysate from the top half of the liquid to ensure that no lysis particles are transferred from the lysis tube to the PCR tube as this can inhibit PCR from occurring—also be sure not to touch the pellet when adding the lysate. Seal the PCR tubes with the flat optical PCR caps provided with the kit. Ensure that the tubes are properly sealed by pressing down firmly over each opening. Do not use the capping tool to seal the PCR tubes. Mix the contents of all the PCR tubes thoroughly by vortexing for 10–15 s to ensure that the pellet is fully rehydrated. Spin the PCR tubes using a plate spinner for at least 10 s to remove bubbles and to collect the liquid at the bottom of the tube before placing in the PCR instrument. Start the run within 30 min of adding sample lysates to the PCR tubes. ( k )Open the instrument drawer. Use the MicroAmp 96-Well Tray for VeriFlex Block to transfer the tubes to the instrument in the same configuration as the plate layout in the software, and then close the instrument drawer. ( l )In the “Run” tab of the experiment file in RapidFinder Analysis Software, select the instrument’s serial number from the Instrument drop-down list. ( m )Click “Start Run,” and then follow the software prompts. PCR Setup—7500 Fast Instrument Use the RapidFinder Express Software to determine the Run Layout (plate layout) for samples based on the information entered and create a run file. Refer to the online help in the software for more details. On the main page of RapidFinder Express Software, select “Create/Edit a Run File,” and then “enter” or “edit” the Run File information at the prompts. Plate layouts can be manually customized in the software. Select “V. cholerae Multiplex SureTect 1.0 or later” for the assay. Following the plate layout previously set up in the software, place the required number of SureTect Vibrio cholerae, V. parahaemolyticus, and V. vulnificus PCR Tubes (PCR tubes) in a suitable rack with a PCR carry plate, and then tap the rack of tubes on the bench to ensure that the pellets are located at the bottom of the tubes. If required by the plate layout, place empty SureTect PCR tubes in the rack to balance the tray when the tubes are placed in the instrument. Allow the PCR tubes to remain on the bench for approximately 5 min, to bring to room temperature, and then open one strip of PCR tubes by removing the seal. PCR pellets are pale yellow. If the pellet has collapsed or is not pale yellow, do not use. If the pellet is not positioned at the bottom of a tube, gently move the pellet to the bottom of the tube with a sterile, empty pipet tip. Do not use a tip containing lysate. Uncap the lysis tubes using the decapping tool. Transfer 20 μL of the lysate or mock-purified sample (negative extraction control reaction) to the appropriate PCR tube to rehydrate the pellet. Tap the rack to ensure that the lysate is at the bottom of the tube and touching the pellet. Remove lysate from the top half of the liquid to ensure that no lysis particles are transferred from the lysis tube to the PCR tube as this can inhibit PCR from occurring. Do not touch the pellet when adding the lysate. Seal the PCR tubes with the flat optical PCR caps provided with the kit. Ensure that the tubes are properly sealed by pressing down firmly over each opening. Do not use the capping tool to seal the PCR tubes. Mix the contents of all PCR tubes thoroughly by vortexing for 10–15 s to ensure that the pellet is fully rehydrated. Spin the PCR tubes using a plate spinner for at least 10 s to remove bubbles and to collect the liquid at the bottom of the tube before placing in the PCR instrument. Start the PCR run within 30 min of adding sample lysates to the PCR tubes. ( j )In the RapidFinder Express Software, select Start Instrument Run on the main page, select the appropriate run file, and follow the software prompts. ( k )Use the PCR carry plate to transfer the tubes to the instrument in the same configuration as the run layout. Use the Precision Plate Holder for SureTect assays. Load empty SureTect PCR tube strips as directed by the software. ( l )Close the tray to the instrument and follow the RapidFinder Express Software prompts to start the run. 

Equilibrate the Lysis Reagent 1 Tubes to room temperature (23.0 ± 5°C). Place the required number of Lysis Reagent 1 Tubes in a MicroAmp Splash-Free 96-Well Base and MicroAmp 96-Well Tray. Ensure that the liquid is collected at the bottom of each tube—flick the tubes in a downward motion or tap the tubes against the bench. Allow the tubes to remain at room temperature for approximately 10 min before opening. Remove the foil from each Lysis Reagent 1 Tube, and then add 10 μL of Proteinase K to the tube. Avoid contamination of the Proteinase K stock tube by using a new filtered pipet tip each time Proteinase K is withdrawn from the stock tube. Use a 10–100 μL repeat pipettor to reduce the number of tips required. Transfer 10 μL of the enriched sample to a lysis tube. For the negative extraction controls, transfer 10 μL of sterile enrichment media to a lysis tube. Ensure that the pipet tip reaches the bottom of the lysis tube to facilitate complete mixing of the sample with Lysis Reagent 1. Seal the tubes with the domed Lysis Tube Caps using the Thermo Scientific PCR Capping Tool or with the Thermo Scientific 96-Well Pierceable Seal using the MicroAmp Adhesive Film Applicator. To conduct the lysis procedure, incubate the samples in the Applied Biosystems SimpliAmp Thermal Cycler set to the parameters outlined in . Note: To prevent crushing of the tubes in the SimpliAmp Thermal Cycler, use the bottom piece from the MicroAmp 96-Well Tray/Retainer Set, or include at least four complete SureTect lysis tube strips. 

Use the RapidFinder Analysis Software to determine the Run Layout (plate layout) for the samples based on the information entered and create a run file. Refer to the online help in the software for more details. On the home page of the RapidFinder Analysis Software, select “Create Experiment,” and then “enter” or “edit” the well parameters. Select Vibrio Multiplex SureTect QS5 version 1.0 (or later) for the assay. Following the plate layout previously set up in the software, place the required number of SureTect Vibrio cholerae, V. parahaemolyticus, and V. vulnificus PCR Tubes (PCR tubes) in the MicroAmp 96-Well Tray for VeriFlex Block. Place the block on the MicroAmp Splash-Free 96-Well Base. Firmly press the PCR tubes to the tray, and then tap the tubes on the bench to ensure that the pellets are located at the bottom of the tubes. Allow the PCR tubes to remain on the bench for approximately 5 min, to bring to room temperature, and then open one strip of PCR tubes by removing the seal. PCR pellets are pale yellow. If the pellet has collapsed or is not pale yellow, do not use. If the pellet is not positioned at the bottom of a tube, gently move the pellet to the bottom of the tube with a sterile, empty pipet tip. Do not use a tip containing lysate. Uncap the lysis tubes using the decapping tool. Transfer 20 μL of the lysate or mock-purified sample (negative extraction control reaction) to the appropriate PCR tube to rehydrate the pellet. Tap the rack to ensure that the lysate is at the bottom of the tube and touching the pellet. Remove lysate from the top half of the liquid to ensure that no lysis particles are transferred from the lysis tube to the PCR tube as this can inhibit PCR from occurring—also be sure not to touch the pellet when adding the lysate. Seal the PCR tubes with the flat optical PCR caps provided with the kit. Ensure that the tubes are properly sealed by pressing down firmly over each opening. Do not use the capping tool to seal the PCR tubes. Mix the contents of all the PCR tubes thoroughly by vortexing for 10–15 s to ensure that the pellet is fully rehydrated. Spin the PCR tubes using a plate spinner for at least 10 s to remove bubbles and to collect the liquid at the bottom of the tube before placing in the PCR instrument. Start the run within 30 min of adding sample lysates to the PCR tubes. ( k )Open the instrument drawer. Use the MicroAmp 96-Well Tray for VeriFlex Block to transfer the tubes to the instrument in the same configuration as the plate layout in the software, and then close the instrument drawer. ( l )In the “Run” tab of the experiment file in RapidFinder Analysis Software, select the instrument’s serial number from the Instrument drop-down list. ( m )Click “Start Run,” and then follow the software prompts. 

Use the RapidFinder Express Software to determine the Run Layout (plate layout) for samples based on the information entered and create a run file. Refer to the online help in the software for more details. On the main page of RapidFinder Express Software, select “Create/Edit a Run File,” and then “enter” or “edit” the Run File information at the prompts. Plate layouts can be manually customized in the software. Select “V. cholerae Multiplex SureTect 1.0 or later” for the assay. Following the plate layout previously set up in the software, place the required number of SureTect Vibrio cholerae, V. parahaemolyticus, and V. vulnificus PCR Tubes (PCR tubes) in a suitable rack with a PCR carry plate, and then tap the rack of tubes on the bench to ensure that the pellets are located at the bottom of the tubes. If required by the plate layout, place empty SureTect PCR tubes in the rack to balance the tray when the tubes are placed in the instrument. Allow the PCR tubes to remain on the bench for approximately 5 min, to bring to room temperature, and then open one strip of PCR tubes by removing the seal. PCR pellets are pale yellow. If the pellet has collapsed or is not pale yellow, do not use. If the pellet is not positioned at the bottom of a tube, gently move the pellet to the bottom of the tube with a sterile, empty pipet tip. Do not use a tip containing lysate. Uncap the lysis tubes using the decapping tool. Transfer 20 μL of the lysate or mock-purified sample (negative extraction control reaction) to the appropriate PCR tube to rehydrate the pellet. Tap the rack to ensure that the lysate is at the bottom of the tube and touching the pellet. Remove lysate from the top half of the liquid to ensure that no lysis particles are transferred from the lysis tube to the PCR tube as this can inhibit PCR from occurring. Do not touch the pellet when adding the lysate. Seal the PCR tubes with the flat optical PCR caps provided with the kit. Ensure that the tubes are properly sealed by pressing down firmly over each opening. Do not use the capping tool to seal the PCR tubes. Mix the contents of all PCR tubes thoroughly by vortexing for 10–15 s to ensure that the pellet is fully rehydrated. Spin the PCR tubes using a plate spinner for at least 10 s to remove bubbles and to collect the liquid at the bottom of the tube before placing in the PCR instrument. Start the PCR run within 30 min of adding sample lysates to the PCR tubes. ( j )In the RapidFinder Express Software, select Start Instrument Run on the main page, select the appropriate run file, and follow the software prompts. ( k )Use the PCR carry plate to transfer the tubes to the instrument in the same configuration as the run layout. Use the Precision Plate Holder for SureTect assays. Load empty SureTect PCR tube strips as directed by the software. ( l )Close the tray to the instrument and follow the RapidFinder Express Software prompts to start the run. 

For the QuantStudio 5 PCR Instrument: Data analysis is automated by the software. For detailed instructions and options for reporting, exporting, and storing results, see the Help function in the software. In the home screen of the RapidFinder Analysis Software, click Results, and then click the sub-tab for the desired view of the data. Summary—plate format Results—table format Details—amplification plot For the 7500 Fast PCR Instrument: Data analysis is automated by the software. In the RapidFinder Express Software, select View Results on the main page, select the appropriate run file, and follow the prompts to view results. To display a list of results in table format, click Table View. Select a sample, and then click View Details to see replicate information about samples. 

Samples with positive PCR results must be confirmed by selective plating and further confirmed by using conventional tests described in the methods standardized by CEN or ISO (EN ISO 21872–1:2017) or BAM Chapter 9 from colonies (including the purification step). The confirmation step must start from the primary enrichment broth. To confirm presumptive positives using the alternative candidate confirmation method: Streak 10 μL of the enriched sample onto plates of TCBS agar and/or CHROMagar Vibrio, Modified mCPC agar, or CC agar. Incubate the plates at 34–38°C for 21–27 h under aerobic conditions. Pick and purify up to five isolated colonies using plates of nonselective saline nutrient agar. Biochemically confirm the colonies using test kits or one of the reference methods. 

This validation study was conducted under the AOAC Research Institute Performance Tested Methods SM Program and the AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces, Appendix J ( ). Method developer studies were conducted in the laboratories of Thermo Fisher Scientific (Basingstoke, UK) and Thermo Fisher Scientific Oy (Vantaa, Finland) and included the inclusivity/exclusivity study and product consistency and stability study. The independent laboratory study was conducted by Mérieux NutriSciences Silliker ® Food Science Center (Crete, IL) and included a matrix study for all five claimed seafood matrixes and a robustness study. The studies were conducted according to the AOAC guidelines for validation of microbiological methods following an unpaired study design. Reference Materials/Strains Research and Development Culture Collection (RDCC) .—Thermo Fisher Scientific, Microbiology Division, Basingstoke, UK. Trials Culture Collection (TCC) .—Thermo Fisher Scientific, Microbiology Division, Basingstoke, UK. National Collection of Type Cultures (NCTC) .—Salisbury, UK. American Type Culture Collection (ATCC) .—Manassas, VA. Culture Collection University of Gothenburg (CCUG) .—Göteborg, Sweden. Research and Development Culture Collection 2 (MH). —Thermo Fisher Scientific, Microbiology Division, Basingstoke, UK. Research and Development Culture Collection 3 (VM/VP). —Thermo Fisher Scientific, Microbiology Division, Basingstoke, UK. Spanish Type Culture Collection (CECT). —Valencia, Spain. Czech Collection of Microorganisms (CCM). —Kralovopolska, Czech Republic. Method Developer Studies Inclusivity and exclusivity study A total of 155 Vibrio inclusivity isolates consisting of 54 V. cholerae , 50 V. parahaemolyticus, and 51 V. vulnificus strains were analyzed with the SureTect Vibrio PCR Assay Method on the 7500 Fast and QuantStudio 5 PCR Instruments. Isolates were obtained from both internal and external research and development culture collections with the origin of strains representing a wide range of food, clinical, and environmental sources. Methodology. —Inclusivity testing was conducted by removing isolates from −80°C storage and culturing each strain on a tryptone soy agar plate overnight. Colonies were then cultured in 10 mL of single-strength alkaline peptone water (APW) at 35 ± 1°C for 24 h, after which 50 μL was transferred to ASPW and incubated at 35 ± 1°C for 8 h. Each isolate was diluted, if necessary, in sterile enrichment media to approximately 10–100 times the LOD 50 of the SureTect Vibrio PCR Assay for testing. A total of 50 exclusivity isolates, consisting of closely related non-target bacterial species and non-target Vibrio species, were analyzed on the 7500 Fast and QuantStudio 5 PCR Instruments. Isolates were obtained from internal and external culture collections. Exclusivity testing was conducted by removing isolates from −80°C storage, streaking onto tryptic soy agar plates, and then incubating at 18–24 h at 37 ± 1°C. Each isolate was then inoculated into tryptone soy broth and incubated for 20–24 h and 37 ± 2°C. Inclusivity and exclusivity isolates were blind-coded and randomized to ensure unbiased processing of samples. Results. —All isolates tested were successfully detected or excluded on both instruments. The results for inclusivity and exclusivity are shown in and , respectively. Product consistency (lot-to-lot) and stability studies The product consistency and stability study examined three lots of the SureTect Vibrio PCR Assay for lot-to-lot variation and product stability to confirm that the manufacturing and performance of the kits were consistent at different points during shelf life. The three kits tested consisted of one lot near the expiration date (13 months), one near the middle of the expiration period (7 months), and one lot that had been recently manufactured (1 month). Methodology. —A strain of V. parahaemolyticus NCTC 10903 was cultured in ASPW at 35.0 ± 1°C for 8 h to act as the target organism for comparison between the three kit lots. This culture was then diluted in sterile enrichment buffer to the limit of detection (LOD 50 ) of the SureTect Vibrio PCR Assay to achieve fractional recovery. A strain of Vibrio fluvialis NCTC 11327 was cultured in ASPW at 35.0 ± 1°C for 22 h to act as the non-target organism for comparison between the kit lots and was not diluted. Each kit lot was tested with 10 replicates each of the target V. parahaemolyticus strain and non-target V. fluvialis strain in a randomized blind-coded fashion. Results. —POD values and CIs were calculated from the data to determine any statistical variation between lots. All V. fluvialis samples were negative for all replicates for all three kits. Fluorescence levels for V. parahaemolyticus were comparable between kit lots with no significant difference seen between the kits at the 5% confidence level after POD analysis. The POD analysis of the stability study is outlined in . Independent Laboratory Studies Matrix study The matrix study was conducted according to the AOAC guidelines Appendix J following an unpaired study design for 50 g of raw tuna, 50 g of raw mussels, 50 g of salmon roll with cream cheese, 50 g of green-lipped mussel extract, and 125 g of cooked shrimp. The SureTect Vibrio PCR Assay method was compared to the qualitative version of the BAM Chapter 9 reference method for all matrixes and additionally to the ISO 21872–1:2017 reference method for raw tuna and raw mussels. The BAM Chapter 9 reference method followed the detection principle as indicated for V. cholerae as opposed to the MPN procedure for V. parahaemolyticus and V. vulnificus to allow for comparison to the candidate nonquantitative detection method. Since MPN methods are based on a series of qualitative methods, it was determined that using the qualitative method found in the BAM MPN to assess performance of the candidate method was appropriate. The method used the same enrichment as detailed in BAM Chapter 9, and the sample enrichment ratio was the same as outlined in BAM Chapter 1, section G.1 ( ). This allowed for direct comparison of the candidate qualitative method to the FDA reference methodology for detection while adhering to determined enrichment procedures and ratios. In addition, a candidate confirmation procedure was included in the study as previously described in the “Confirmation” section. Methodology. —Raw tuna, raw mussels, salmon roll with cream cheese, green-lipped mussel extract, and cooked shrimp were purchased from a local supplier and prescreened for natural contamination of V. cholerae, V. parahaemolyticus, and V. vulnificus following the candidate method. The mesophilic aerobic plate count (APC) was determined following the BAM Chapter 3 Aerobic Plate Count reference method ( ). No natural contamination was detected in the screening of all five products; however, late amplification was seen in the cooked shrimp matrix and one batch of the green-lipped mussel extract, indicating potential low-level contamination or the presence of dead cells. The presence of dead cells rather than viable low-level contamination was determined by nonselective and selective plating of the enrichment, as well as by screening of the matrix at 0 and 16 h and a comparison of PCR cycle threshold (C t ) values. The C t value did not change between the timepoints. Had there been low-level contamination present, the C t value would have decreased (i.e., 36 to 27 due to growth of the contaminant during incubation). However, as the C t value did not change and growth was not recoverable on any agar plate, the PCR amplification seen during screening was most likely due to dead cell DNA. This issue was addressed by performing a 1-in-10 dilution after enrichment that removed the dead cell amplification (all samples had very late C t values—greater than 35), as described in the troubleshooting steps in the instructions for use should an end user encounter this problem. The matrix study consisted of evaluating a total of thirty 50 g portions of raw tuna, raw mussels, salmon roll with cream cheese, and green-lipped mussel extract and 125 g portions of cooked shrimp following an unpaired study design. Within each sample set, there were five non-inoculated portions (POD = 0), 20 low-level inoculated portions (targeting POD = 0.25–0.75), and five high-level inoculated portions (targeting POD = 1). For the raw tuna, raw mussels, and salmon roll with cream cheese, inoculation was conducted with a liquid culture. Raw tuna was inoculated with V. cholerae , raw mussels were inoculated with V. parahaemolyticus , and salmon roll with cream cheese was inoculated with all three target species. These matrixes were artificially contaminated as follows. For the raw tuna, V. cholerae was grown in ASPW for 24 h at 35 ± 1°C, and then serially diluted with Dulbecco’s Phosphate Buffered Saline (DPBS) and plated onto TSA with 2% NaCl. The culture level was determined and the amount of inoculum to add to the matrix was calculated based on the target level. The raw tuna was cut, placed into a large sterile bag, and homogenized by hand-mixing. Low- and high-level contaminated tuna was prepared by weighing the appropriate amount of tuna into a bag and inoculating with a dilution of V. cholerae to obtain fractional recovery for the low level and all positives for the high level. The bags of inoculated raw tuna were stored at 2–8°C for 72 h. After storage, 25 g of inoculated tuna was combined with 25 g of non-inoculated tuna to create the 50 g test portion on the day of analysis. For the raw mussels, V . parahaemolyticus was grown in ASPW for 24 h at 35 ± 1°C, and then serially diluted with DPBS and plated onto TSA with 2% NaCl. The culture level was determined and the amount of inoculum to add to the matrix was calculated based on the target level. The raw mussels were removed from the shell, cut up, placed in a large sterile bag, and homogenized by hand-mixing. Low- and high-level contaminated mussels were prepared by weighing the appropriate amount into a bag and inoculating with a dilution of V. parahaemolyticus to obtain fractional recovery in the low level and all positives in the high level. The bags of inoculated raw mussels were stored at 2–8°C for 72 h. After storage, a 25 g portion was removed and combined with 25 g of non-inoculated mussels to create the 50 g test portion on the day of analysis. For the salmon roll with cream cheese, each target strain ( V. cholerae, V . parahaemolyticus, and V. vulnificus ) was grown in ASPW for 24 h at 35 ± 1°C and then serially diluted with DPBS and plated onto TSA with 2% NaCl. The culture level was determined and the amount of inoculum to add to the matrix was calculated based on the target level. The salmon roll was cut up, transferred to a large sterile bag and homogenized with cream cheese by hand mixing; the bag contained 70% salmon and 30% cream cheese. Low- and high-level contaminated materials were prepared by weighing the appropriate amount of homogenized salmon roll into a bag and inoculating with a dilution of each strain to obtain fractional recovery in the low level and all positives in the high level. The bags of inoculated salmon roll with cream cheese were stored at 2–8°C for 72 h. After storage, a 25 g portion was removed and combined with 25 g of non-inoculated material to create the 50 g test portion on the day of analysis. For the green-lipped mussel extract, inoculation was conducted with a lyophilized culture of V. parahaemolyticus . The culture was grown in 400 mL of ASPW for 24 h at 35.0 ± 1.0°C and pelleted by centrifugation. Four grams of gelatin, 5 g NaCl, and 25 g skimmed milk were mixed in 225 mL of phosphate buffer, 10 mL of which was then added to the spun-down culture pellet and vortexed until the pellet fully dissolved. The 10 mL of dissolved pellet was transferred to a glass flask and then placed in a −80°C freezer for 30 min. After 30 min, the glass flask was placed in a freeze-dryer for 24 h. After 24 h, the glass flask was removed, and the dried culture was crushed into a fine powder. The fine powder was added to 200 g of green-lipped mussel extract to make the seed culture. The seed remained at room temperature (18–25°C) for 48 h before being diluted with uninoculated green-lipped mussel extract. Typically, room temperature stable products must equilibrate for 2 weeks before testing, but because the Vibrio inoculum would not survive that long, AOAC agreed that the equilibration time could be reduced to 48 h. High and low contamination levels were prepared using the diluted seed culture and homogenized. After storage, a 25 g portion was removed and combined with 25 g of non-inoculated material to create the 50 g test portion on the day of analysis. For the cooked shrimp, a liquid heat-stressed culture of V. vulnificus was used. The culture was grown in ASPW for 48 h at 35 ± 1°C and then serially diluted in DPBS. The culture was heat-stressed at 46°C for 50 s, and then plated onto TSA with and without 2% NaCl. Percentage of injury was determined by counting the number of colonies on the selective agar, dividing by the number of colonies on the nonselective agar, subtracting from 1, and then multiplying by 100 (50–80% injury was required). The inoculating culture was found to have an injury level of 77%. Shrimp were cut up, transferred to a large sterile bag using sterile scissors, and then hand-homogenized. A low-level and high-level bag of cooked shrimp was weighed and then inoculated with a specific target level. The inoculated cooked shrimp were then stored at 4°C for 48 h. After storage, a 25 g portion was removed and combined with 100 g of non-inoculated material to create the 125 g test portion on the day of analysis. BAM Chapter 9 Method. —The BAM Chapter 9 reference method was followed as described for V. cholerae , in agreement with AOAC to detect all Vibrio species. The 25 g test portions were weighed into a tared jar and enriched with 225 mL of APW and then mixed thoroughly for 2 min. Test portions were incubated for 6–8 h at 35 ± 2°C and reincubated overnight if the sample had been processed (cooked shrimp, green-lipped mussel extract). After incubation, a 3 mm loopful of the surface pellicle of the APW culture was streaked onto TCBS and mCPC or CC agar plates. The TCBS plates were incubated for 18–24 h at 35 ± 2°C, and the mCPC or CC plates were incubated for 18–24 h at 39–40°C. Following incubation, the plates were examined for typical colony morphology of the target organism. Typical colonies of V. cholerae on TCBS agar are large (2–3 mm), smooth, yellow, and slightly flattened with opaque centers and translucent peripheries. Typical colonies of V. cholerae on mCPC or CC agar are small, smooth, opaque, and green to purple in color, with a purple background on extended incubation. Typical colonies of V. parahaemolyticus on TCBS agar are round, opaque, green, or bluish. Most strains of V. parahaemolyticus will not grow on mCPC or CC agar. If growth occurs, colonies will be green-purple in color due to lack of cellobiose fermentation. Typical colonies of V. vulnificus on TCBS agar are round, opaque, green, or bluish, and typical colonies of V. vulnificus on mCPC or CC agar are round, flat, opaque, yellow, and 1–2 mm in diameter. A minimum of two suspect colonies were picked for confirmation and streaked onto TSA with 2% NaCl and incubated at 35 ± 2°C for 18–24 h. After incubation, one colony per sample was suspended in 0.85% NaCl and confirmed using an API 20E kit. ISO 21872–1:2017 Method. —For the ISO 21872–1:2017 reference method, a 25 g test portion was homogenized in 225 mL of ASPW and incubated for 6 ± 1 h at 41.5 ± 1.0°C. After 6 h, 1 mL of the enriched sample was transferred into a tube containing 10 mL of ASPW and incubated at 41.5 ± 1.0°C for 18 ± 1 h. After incubation, 1 μL of sample was streaked onto TCBS and mCPC or CC agar. The TCBS plates were incubated at 37.0 ± 1.0°C for 24 ± 1 h, and the mCPC or CC agar was incubated at 39–40°C for 24 ± 1 h. After incubation, the plates were examined for typical colony morphology of the target strains ( see “BAM Chapter 9 Method” for colony morphology descriptions). A minimum of two suspect colonies were picked for confirmation and streaked onto Tryptone Soy Agar with 2% NaCl and incubated at 35.0 ± 2.0°C for 18–24 h. After incubation, one colony per sample was suspended in 0.85% NaCl and confirmed using an API 20E kit. The Thermo Scientific SureTect Vibrio cholerae, V. parahaemolyticus, and V. vulnificus PCR Assay. —All test portions were analyzed by following the SureTect Vibrio PCR Assay workflow after 8–20 h of enrichment for salmon roll with cream cheese and 16 h ± 10 min of enrichment for the remaining matrixes. All test portions were analyzed using the Applied Biosystems QuantStudio 5 Real-Time PCR Instrument and 7500 Fast Real-Time PCR Instrument. All portions, regardless of presumptive results, were culturally confirmed according to the BAM Chapter 9 reference method for all matrixes and the ISO 21872–1:2017 reference method for raw tuna and raw mussels. In addition, a candidate confirmation was performed as previously described in the section “Confirmation.” Most Probable Number Analysis. —For the BAM Chapter 9 method, the concentration of V. cholerae, V. parahaemolyticus, and V. vulnificus in the low-level inoculum for all 25 g test portions was determined by Most Probable Number (MPN) on the day of analysis by evaluating 20 × 25 g (BAM Ch. 9 reference method test portions), 5 × 50 g, and 5 × 10 g uninoculated portions. The concentration for each strain in the high-level inoculum was determined by MPN by evaluating 5 × 25 g (BAM Ch. 9 reference method test portions), 5 × 10 g, and 5 × 5 g inoculated test portions. For the 50 g portions 450 mL of APW was added, for the 10 g portions 90 mL of APW was used, and for the 5 g portions 45 mL of APW was used. For the ISO 21872–1:2017 reference method, the concentration of each target strain in the low-level inoculum for all test portions was determined by MPN on the day of analysis by evaluating 20 × 25 g (reference method test portions), 5 × 50 g, and 5 × 10 g uninoculated portions. For the high inoculum level, the concentration of each strain was determined by MPN by evaluating 5 × 25 g (refence method test portions), 5 × 10 g, and 5 × 5 g inoculated portions. For the 50 g portions 450 mL of APW was added, for the 10 g portions 90 mL of APW was used, and for the 5 g portions 45 mL of APW was used. The number of positives from the three test levels was used to calculate the MPN using the Least Cost Formulation Most Probable Number calculator, version 1.6 (Virginia Beach, VA) provided by AOAC RI. Results. —As per criteria outlined in Appendix J, fractional positive results were obtained for 50 g test portions of raw tuna, raw mussels, salmon roll with cream cheese, green-lipped mussel extract, and cooked shrimp for the SureTect Vibrio PCR Assay method. Prior to inoculation, an APC result of 3.9 × 10 2 cfu/g was established for cooked shrimp, 2.0 × 10 1 cfu/g for raw mussels, 9.1 × 10 3 cfu/g for salmon roll with cream cheese, 8.8 × 10 3 cfu/g for raw tuna, and 0 cfu/g for green lipped mussel extract. For the high spike level for the candidate and both reference methods, the number of positives detected was slightly below AOAC and ISO requirements. During preparation of inoculating culture, numerous attempts were made to achieve a higher spiking level, but unfortunately the number of positives detected at the higher level fell below the stipulated requirements. The candidate method detected 3/5 positives or above for all matrixes tested with a POD c of 0.60 or higher, compared to the reference methods, which detected anywhere from 0 to 5/5 positives for the high level. This is likely due to stress caused by the required fridge hold/heat-stress/desiccation (depending on matrix) required prior to analysis. Vibrio spp. are well documented for undergoing genotypic and, consequently, phenotypic changes when under stress (particularly cold stress) to ensure survival through alteration of different metabolic pathways, which then leads to a lag in growth phase when transferred to normal growth conditions and typical pathways must be upregulated again ( ). A study looking at temperature upshift in V. parahaemolyticus after cold stressed found it took up to almost 48 h for cells to return to typical growth (normal cellular shape and upregulation of certain metabolic pathways, for example) ( ). This is likely why the Vibrio strains had recovered to a fractional level at the time of analysis but did not meet the higher spike level target. Evidence for this can also be seen when looking at the individual matrixes. For the processed GLME and cooked shrimp matrixes, the candidate method had identical performance to the BAM reference method, and both methods hit the stipulated spike level of 5/5 positives. This is likely because the incubation timepoints were more comparable 18–24 h for BAM and 16 h for candidate (BAM method stipulates reincubation for processed matrixes, as mentioned in the previous section) between the reference methods and longer compared to the unprocessed matrixes, which allowed a longer time for stressed cell recovery. However, for the non-processed matrixes (raw tuna, raw mussels, and salmon roll with cream cheese), where the reference method timepoints were substantially shortened (6–8 h for BAM compared to 18–24 h for the processed matrixes), there was a notable difference in high spike positives detected. The candidate method detected 3/5 positives at the high level for all matrixes compared to 0/5, 2/5, and 1/5 for the BAM method, respectively. The candidate method timepoints were 16 h for both raw tuna and raw mussels, and for salmon roll with cream cheese it was 8 h; however, prewarmed media is used to support cell recovery. For the ISO reference method, even though the timepoint was 18–24 h, there is the added complication of higher background flora associated with raw tuna ( ), which can cause difficulties in recovering cells due to competition between species. The ISO data were more comparable to the candidate data, however, 2/5 vs 3/5 positives, respectively, likely due to the slightly longer timepoint. Overall, this demonstrates that the shorter incubation did not allow enough time for the Vibrio to recover to the levels anticipated in a higher spike. Furthermore, the performance of the matrixes would suggest that the genotypic and phenotypic changes seen within Vibrio in stress-inducing situations appear to be more severe in cold environments compared to heat and desiccation, as recovery in GLME and cooked shrimp was far more substantial than the matrixes that underwent a fridge hold. For the candidate method, the fractional spike levels were hit for all matrixes, and while the high level did not hit 5/5 positives for every matrix, as there were no issues with detection of a lower spike level this lends confidence to the ability of the assay to detect higher cell loads; therefore, the lower positives achieved here should not warrant concern for performance of the assay. The POD was calculated as the number of positive outcomes divided by the total number of trials using the Least Cost Formulations, Ltd, AOAC Binary Data Interlaboratory Study Workbook version 5.2 (Virgina Beach, VA) ( ). The POD was calculated for the candidate presumptive results, POD CP , the candidate confirmatory results, POD CC , the difference in the candidate presumptive and confirmatory results, dPOD CP, presumptive candidate results that confirmed positive, POD C, the reference method, POD R , and the difference in the confirmed candidate and reference methods, dPOD C . The POD analysis between the SureTect Vibrio PCR Assay and reference methods indicated that there was no significant difference at the 5% confidence level between the number of positive results by the methods for raw mussels, green-lipped mussel extract, and cooked shrimp matrixes for the BAM method and the raw tuna and raw mussel matrixes for the ISO method. For raw tuna and the salmon roll with cream cheese, POD analysis showed superior performance of the candidate method due to poor performance of the BAM reference method. For raw tuna, the SureTect Vibrio PCR Assay detected eight confirmed positives for the low level compared to zero positives detected by the BAM method; this was also the case for the high level, with three positives confirmed from the SureTect Vibrio PCR Assay compared to zero from the BAM method. For the salmon roll with cream cheese, the SureTect Vibrio PCR Assay detected nine confirmed positives for the low level compared to two for the BAM method. For the high level, although not statistically significant, the SureTect Vibrio PCR Assay detected three confirmed positives compared to just one with the BAM method. In general, although not statistically significant for all matrixes, the data show a trend of higher detection for the SureTect Vibrio PCR Assay compared to the BAM method. The POD analysis between the SureTect Vibrio PCR Assay method presumptive and confirmed results indicated no significant difference at the 5% level for all methods following both reference confirmation method procedures and the candidate method confirmation procedure except for raw tuna, in which 18 presumptive positives were detected compared to eight confirmed using the reference confirmation. A summary of the POD analyses is presented in – . Salmon roll with cream cheese: Further investigative study and results. —The SureTect Vibrio PCR assay is a multiplex PCR assay and therefore can be used to detect all three target strains of V. cholerae, V. parahaemolyticus, and V. vulnificus from one single sample whether one, two, or all three target strains are present. As such, the initial study design was to triple-infect the salmon roll with cream cheese as outlined previously to demonstrate the multiplex capability of the assay. However, it is well documented that multiple Vibrio spp. exhibit both inter- and intra-specific competition to occupy and thrive within the same ecological niche ( ). For example, both V. parahaemolyticus and V. cholerae use the well-characterized type VI secretory system to compete with other bacterial species, other Vibrio spp., and different strains of V. cholerae and V. parahaemolyticus by secreting anti-eukaryotic and prokaryotic effectors ( , ). The geographical spread (origin of the sample) and seasonality of each target strain also need to be considered. For example, populations of V. parahaemolyticus in the Pacific Northwest of the US peak in June but then wane throughout the rest of the year ( ). This contrasts with the coast of South Korea, where V. parahaemolyticus remains ubiquitous all year round compared to V. cholerae and V. vulnificus , which are seasonally affected and isolated specifically during the summer months ( ). The inherent competition, geographical spread, and seasonality between the different strains mean it is unlikely to see a sample that is dual or triple infected, but not impossible. If this is the case, further difficulty is encountered when trying to isolate and confirm suspect colonies on culture media due to the differing growth rates between the target strains. During the salmon roll with cream cheese matrix study, there was PCR detection and amplification of the three spiked strains, but considerable difficulty in confirming these on agar. Looking at the generated PCR C t values showed a 1–2 log difference in growth of the target strains as anticipated due to the differing growth rates between targets. Therefore, this 1–2 log difference had likely caused overgrowth of the higher-density strain on the plate, which negated confirmation of the other species present. A multiplex study was devised to demonstrate the capability of the SureTect Vibrio PCR Assay method to detect all three target strains and to also demonstrate the difficulties encountered in isolation and confirmation of all three target species when present at different growth levels. The study looked at several combinations of all three target organisms with a 1–2 log difference compared to a combination where each target was spiked at the same level, and an uninoculated/screening level in salmon roll with cream cheese. All combinations were spiked post-enrichment to negate the effect of the different growth rates and run on the QuantStudio 5 PCR Instrument; all combinations were then plated onto TCBS, CHROMagar Vibrio, and mCPC agar to assess recovery. All combinations are outlined in , and agar scoring is outlined in . The results of the study were as expected. When the difference between the strains was 2-log growth, recovery of all three target strains was not possible on agar despite positive detection by PCR. When the difference was 1-log, it was possible to isolate two of the three target strains, and when all three target strains were at the same growth level, all three were recoverable on agar. The decision was made to use only the V. parahaemolyticus data in this matrix and not to continue with triple inoculation. The results of the multiplex spike study are outlined in and and . Robustness study For the robustness study, the SureTect Vibrio PCR Assay method was evaluated by adjusting three testing parameters. The three parameters included: enrichment time (7, 8, and 24 h), volume of sample lysed (9, 10, and 11 μL), and volume of lysate (18, 20, and 22 μL). A total of nine combinations were evaluated, with the ninth combination prepared as nominal conditions to provide a comparison. Methodology. —Twenty 50 g test portions of salmon roll with cream cheese were analyzed. Ten were inoculated with V. cholerae at a level to yield fractional positive results, and 10 were uninoculated. All 50 g test portions of salmon roll with cream cheese were enriched in 450 ± 10 mL of ASPW and incubated according to the enrichment times summarized in . All portions were randomized and blind-coded. Results. —POD analysis of the performance of the SureTect Vibrio PCR assay method demonstrated no statistically significant differences between the nominal conditions and changes in testing parameters. However, the shorter enrichment time of 7 h using the 7500 Fast or the QuantStudio 5 PCR Instrument impacted the number of positives detected by the SureTect Vibrio Assay. Although the POD analysis showed no statistically significant difference between the nominal conditions and the shorter timepoint as mentioned above, the number of positives detected at 7 and 8 h (1 versus 4/5, respectively, depending on instrument) suggests that additional time for incubation is necessary. Therefore, the assay is robust at the longer incubation timepoint but not at the shorter timepoint, indicating that the sample must be incubated for the minimum time specified. The POD results and 95% confidence intervals for each target analyte and treatment combination are presented in and . 

Research and Development Culture Collection (RDCC) .—Thermo Fisher Scientific, Microbiology Division, Basingstoke, UK. Trials Culture Collection (TCC) .—Thermo Fisher Scientific, Microbiology Division, Basingstoke, UK. National Collection of Type Cultures (NCTC) .—Salisbury, UK. American Type Culture Collection (ATCC) .—Manassas, VA. Culture Collection University of Gothenburg (CCUG) .—Göteborg, Sweden. Research and Development Culture Collection 2 (MH). —Thermo Fisher Scientific, Microbiology Division, Basingstoke, UK. Research and Development Culture Collection 3 (VM/VP). —Thermo Fisher Scientific, Microbiology Division, Basingstoke, UK. Spanish Type Culture Collection (CECT). —Valencia, Spain. Czech Collection of Microorganisms (CCM). —Kralovopolska, Czech Republic. 

Inclusivity and exclusivity study A total of 155 Vibrio inclusivity isolates consisting of 54 V. cholerae , 50 V. parahaemolyticus, and 51 V. vulnificus strains were analyzed with the SureTect Vibrio PCR Assay Method on the 7500 Fast and QuantStudio 5 PCR Instruments. Isolates were obtained from both internal and external research and development culture collections with the origin of strains representing a wide range of food, clinical, and environmental sources. Methodology. —Inclusivity testing was conducted by removing isolates from −80°C storage and culturing each strain on a tryptone soy agar plate overnight. Colonies were then cultured in 10 mL of single-strength alkaline peptone water (APW) at 35 ± 1°C for 24 h, after which 50 μL was transferred to ASPW and incubated at 35 ± 1°C for 8 h. Each isolate was diluted, if necessary, in sterile enrichment media to approximately 10–100 times the LOD 50 of the SureTect Vibrio PCR Assay for testing. A total of 50 exclusivity isolates, consisting of closely related non-target bacterial species and non-target Vibrio species, were analyzed on the 7500 Fast and QuantStudio 5 PCR Instruments. Isolates were obtained from internal and external culture collections. Exclusivity testing was conducted by removing isolates from −80°C storage, streaking onto tryptic soy agar plates, and then incubating at 18–24 h at 37 ± 1°C. Each isolate was then inoculated into tryptone soy broth and incubated for 20–24 h and 37 ± 2°C. Inclusivity and exclusivity isolates were blind-coded and randomized to ensure unbiased processing of samples. Results. —All isolates tested were successfully detected or excluded on both instruments. The results for inclusivity and exclusivity are shown in and , respectively. Product consistency (lot-to-lot) and stability studies The product consistency and stability study examined three lots of the SureTect Vibrio PCR Assay for lot-to-lot variation and product stability to confirm that the manufacturing and performance of the kits were consistent at different points during shelf life. The three kits tested consisted of one lot near the expiration date (13 months), one near the middle of the expiration period (7 months), and one lot that had been recently manufactured (1 month). Methodology. —A strain of V. parahaemolyticus NCTC 10903 was cultured in ASPW at 35.0 ± 1°C for 8 h to act as the target organism for comparison between the three kit lots. This culture was then diluted in sterile enrichment buffer to the limit of detection (LOD 50 ) of the SureTect Vibrio PCR Assay to achieve fractional recovery. A strain of Vibrio fluvialis NCTC 11327 was cultured in ASPW at 35.0 ± 1°C for 22 h to act as the non-target organism for comparison between the kit lots and was not diluted. Each kit lot was tested with 10 replicates each of the target V. parahaemolyticus strain and non-target V. fluvialis strain in a randomized blind-coded fashion. Results. —POD values and CIs were calculated from the data to determine any statistical variation between lots. All V. fluvialis samples were negative for all replicates for all three kits. Fluorescence levels for V. parahaemolyticus were comparable between kit lots with no significant difference seen between the kits at the 5% confidence level after POD analysis. The POD analysis of the stability study is outlined in . 

A total of 155 Vibrio inclusivity isolates consisting of 54 V. cholerae , 50 V. parahaemolyticus, and 51 V. vulnificus strains were analyzed with the SureTect Vibrio PCR Assay Method on the 7500 Fast and QuantStudio 5 PCR Instruments. Isolates were obtained from both internal and external research and development culture collections with the origin of strains representing a wide range of food, clinical, and environmental sources. Methodology. —Inclusivity testing was conducted by removing isolates from −80°C storage and culturing each strain on a tryptone soy agar plate overnight. Colonies were then cultured in 10 mL of single-strength alkaline peptone water (APW) at 35 ± 1°C for 24 h, after which 50 μL was transferred to ASPW and incubated at 35 ± 1°C for 8 h. Each isolate was diluted, if necessary, in sterile enrichment media to approximately 10–100 times the LOD 50 of the SureTect Vibrio PCR Assay for testing. A total of 50 exclusivity isolates, consisting of closely related non-target bacterial species and non-target Vibrio species, were analyzed on the 7500 Fast and QuantStudio 5 PCR Instruments. Isolates were obtained from internal and external culture collections. Exclusivity testing was conducted by removing isolates from −80°C storage, streaking onto tryptic soy agar plates, and then incubating at 18–24 h at 37 ± 1°C. Each isolate was then inoculated into tryptone soy broth and incubated for 20–24 h and 37 ± 2°C. Inclusivity and exclusivity isolates were blind-coded and randomized to ensure unbiased processing of samples. Results. —All isolates tested were successfully detected or excluded on both instruments. The results for inclusivity and exclusivity are shown in and , respectively. 

The product consistency and stability study examined three lots of the SureTect Vibrio PCR Assay for lot-to-lot variation and product stability to confirm that the manufacturing and performance of the kits were consistent at different points during shelf life. The three kits tested consisted of one lot near the expiration date (13 months), one near the middle of the expiration period (7 months), and one lot that had been recently manufactured (1 month). Methodology. —A strain of V. parahaemolyticus NCTC 10903 was cultured in ASPW at 35.0 ± 1°C for 8 h to act as the target organism for comparison between the three kit lots. This culture was then diluted in sterile enrichment buffer to the limit of detection (LOD 50 ) of the SureTect Vibrio PCR Assay to achieve fractional recovery. A strain of Vibrio fluvialis NCTC 11327 was cultured in ASPW at 35.0 ± 1°C for 22 h to act as the non-target organism for comparison between the kit lots and was not diluted. Each kit lot was tested with 10 replicates each of the target V. parahaemolyticus strain and non-target V. fluvialis strain in a randomized blind-coded fashion. Results. —POD values and CIs were calculated from the data to determine any statistical variation between lots. All V. fluvialis samples were negative for all replicates for all three kits. Fluorescence levels for V. parahaemolyticus were comparable between kit lots with no significant difference seen between the kits at the 5% confidence level after POD analysis. The POD analysis of the stability study is outlined in . 

Matrix study The matrix study was conducted according to the AOAC guidelines Appendix J following an unpaired study design for 50 g of raw tuna, 50 g of raw mussels, 50 g of salmon roll with cream cheese, 50 g of green-lipped mussel extract, and 125 g of cooked shrimp. The SureTect Vibrio PCR Assay method was compared to the qualitative version of the BAM Chapter 9 reference method for all matrixes and additionally to the ISO 21872–1:2017 reference method for raw tuna and raw mussels. The BAM Chapter 9 reference method followed the detection principle as indicated for V. cholerae as opposed to the MPN procedure for V. parahaemolyticus and V. vulnificus to allow for comparison to the candidate nonquantitative detection method. Since MPN methods are based on a series of qualitative methods, it was determined that using the qualitative method found in the BAM MPN to assess performance of the candidate method was appropriate. The method used the same enrichment as detailed in BAM Chapter 9, and the sample enrichment ratio was the same as outlined in BAM Chapter 1, section G.1 ( ). This allowed for direct comparison of the candidate qualitative method to the FDA reference methodology for detection while adhering to determined enrichment procedures and ratios. In addition, a candidate confirmation procedure was included in the study as previously described in the “Confirmation” section. Methodology. —Raw tuna, raw mussels, salmon roll with cream cheese, green-lipped mussel extract, and cooked shrimp were purchased from a local supplier and prescreened for natural contamination of V. cholerae, V. parahaemolyticus, and V. vulnificus following the candidate method. The mesophilic aerobic plate count (APC) was determined following the BAM Chapter 3 Aerobic Plate Count reference method ( ). No natural contamination was detected in the screening of all five products; however, late amplification was seen in the cooked shrimp matrix and one batch of the green-lipped mussel extract, indicating potential low-level contamination or the presence of dead cells. The presence of dead cells rather than viable low-level contamination was determined by nonselective and selective plating of the enrichment, as well as by screening of the matrix at 0 and 16 h and a comparison of PCR cycle threshold (C t ) values. The C t value did not change between the timepoints. Had there been low-level contamination present, the C t value would have decreased (i.e., 36 to 27 due to growth of the contaminant during incubation). However, as the C t value did not change and growth was not recoverable on any agar plate, the PCR amplification seen during screening was most likely due to dead cell DNA. This issue was addressed by performing a 1-in-10 dilution after enrichment that removed the dead cell amplification (all samples had very late C t values—greater than 35), as described in the troubleshooting steps in the instructions for use should an end user encounter this problem. The matrix study consisted of evaluating a total of thirty 50 g portions of raw tuna, raw mussels, salmon roll with cream cheese, and green-lipped mussel extract and 125 g portions of cooked shrimp following an unpaired study design. Within each sample set, there were five non-inoculated portions (POD = 0), 20 low-level inoculated portions (targeting POD = 0.25–0.75), and five high-level inoculated portions (targeting POD = 1). For the raw tuna, raw mussels, and salmon roll with cream cheese, inoculation was conducted with a liquid culture. Raw tuna was inoculated with V. cholerae , raw mussels were inoculated with V. parahaemolyticus , and salmon roll with cream cheese was inoculated with all three target species. These matrixes were artificially contaminated as follows. For the raw tuna, V. cholerae was grown in ASPW for 24 h at 35 ± 1°C, and then serially diluted with Dulbecco’s Phosphate Buffered Saline (DPBS) and plated onto TSA with 2% NaCl. The culture level was determined and the amount of inoculum to add to the matrix was calculated based on the target level. The raw tuna was cut, placed into a large sterile bag, and homogenized by hand-mixing. Low- and high-level contaminated tuna was prepared by weighing the appropriate amount of tuna into a bag and inoculating with a dilution of V. cholerae to obtain fractional recovery for the low level and all positives for the high level. The bags of inoculated raw tuna were stored at 2–8°C for 72 h. After storage, 25 g of inoculated tuna was combined with 25 g of non-inoculated tuna to create the 50 g test portion on the day of analysis. For the raw mussels, V . parahaemolyticus was grown in ASPW for 24 h at 35 ± 1°C, and then serially diluted with DPBS and plated onto TSA with 2% NaCl. The culture level was determined and the amount of inoculum to add to the matrix was calculated based on the target level. The raw mussels were removed from the shell, cut up, placed in a large sterile bag, and homogenized by hand-mixing. Low- and high-level contaminated mussels were prepared by weighing the appropriate amount into a bag and inoculating with a dilution of V. parahaemolyticus to obtain fractional recovery in the low level and all positives in the high level. The bags of inoculated raw mussels were stored at 2–8°C for 72 h. After storage, a 25 g portion was removed and combined with 25 g of non-inoculated mussels to create the 50 g test portion on the day of analysis. For the salmon roll with cream cheese, each target strain ( V. cholerae, V . parahaemolyticus, and V. vulnificus ) was grown in ASPW for 24 h at 35 ± 1°C and then serially diluted with DPBS and plated onto TSA with 2% NaCl. The culture level was determined and the amount of inoculum to add to the matrix was calculated based on the target level. The salmon roll was cut up, transferred to a large sterile bag and homogenized with cream cheese by hand mixing; the bag contained 70% salmon and 30% cream cheese. Low- and high-level contaminated materials were prepared by weighing the appropriate amount of homogenized salmon roll into a bag and inoculating with a dilution of each strain to obtain fractional recovery in the low level and all positives in the high level. The bags of inoculated salmon roll with cream cheese were stored at 2–8°C for 72 h. After storage, a 25 g portion was removed and combined with 25 g of non-inoculated material to create the 50 g test portion on the day of analysis. For the green-lipped mussel extract, inoculation was conducted with a lyophilized culture of V. parahaemolyticus . The culture was grown in 400 mL of ASPW for 24 h at 35.0 ± 1.0°C and pelleted by centrifugation. Four grams of gelatin, 5 g NaCl, and 25 g skimmed milk were mixed in 225 mL of phosphate buffer, 10 mL of which was then added to the spun-down culture pellet and vortexed until the pellet fully dissolved. The 10 mL of dissolved pellet was transferred to a glass flask and then placed in a −80°C freezer for 30 min. After 30 min, the glass flask was placed in a freeze-dryer for 24 h. After 24 h, the glass flask was removed, and the dried culture was crushed into a fine powder. The fine powder was added to 200 g of green-lipped mussel extract to make the seed culture. The seed remained at room temperature (18–25°C) for 48 h before being diluted with uninoculated green-lipped mussel extract. Typically, room temperature stable products must equilibrate for 2 weeks before testing, but because the Vibrio inoculum would not survive that long, AOAC agreed that the equilibration time could be reduced to 48 h. High and low contamination levels were prepared using the diluted seed culture and homogenized. After storage, a 25 g portion was removed and combined with 25 g of non-inoculated material to create the 50 g test portion on the day of analysis. For the cooked shrimp, a liquid heat-stressed culture of V. vulnificus was used. The culture was grown in ASPW for 48 h at 35 ± 1°C and then serially diluted in DPBS. The culture was heat-stressed at 46°C for 50 s, and then plated onto TSA with and without 2% NaCl. Percentage of injury was determined by counting the number of colonies on the selective agar, dividing by the number of colonies on the nonselective agar, subtracting from 1, and then multiplying by 100 (50–80% injury was required). The inoculating culture was found to have an injury level of 77%. Shrimp were cut up, transferred to a large sterile bag using sterile scissors, and then hand-homogenized. A low-level and high-level bag of cooked shrimp was weighed and then inoculated with a specific target level. The inoculated cooked shrimp were then stored at 4°C for 48 h. After storage, a 25 g portion was removed and combined with 100 g of non-inoculated material to create the 125 g test portion on the day of analysis. BAM Chapter 9 Method. —The BAM Chapter 9 reference method was followed as described for V. cholerae , in agreement with AOAC to detect all Vibrio species. The 25 g test portions were weighed into a tared jar and enriched with 225 mL of APW and then mixed thoroughly for 2 min. Test portions were incubated for 6–8 h at 35 ± 2°C and reincubated overnight if the sample had been processed (cooked shrimp, green-lipped mussel extract). After incubation, a 3 mm loopful of the surface pellicle of the APW culture was streaked onto TCBS and mCPC or CC agar plates. The TCBS plates were incubated for 18–24 h at 35 ± 2°C, and the mCPC or CC plates were incubated for 18–24 h at 39–40°C. Following incubation, the plates were examined for typical colony morphology of the target organism. Typical colonies of V. cholerae on TCBS agar are large (2–3 mm), smooth, yellow, and slightly flattened with opaque centers and translucent peripheries. Typical colonies of V. cholerae on mCPC or CC agar are small, smooth, opaque, and green to purple in color, with a purple background on extended incubation. Typical colonies of V. parahaemolyticus on TCBS agar are round, opaque, green, or bluish. Most strains of V. parahaemolyticus will not grow on mCPC or CC agar. If growth occurs, colonies will be green-purple in color due to lack of cellobiose fermentation. Typical colonies of V. vulnificus on TCBS agar are round, opaque, green, or bluish, and typical colonies of V. vulnificus on mCPC or CC agar are round, flat, opaque, yellow, and 1–2 mm in diameter. A minimum of two suspect colonies were picked for confirmation and streaked onto TSA with 2% NaCl and incubated at 35 ± 2°C for 18–24 h. After incubation, one colony per sample was suspended in 0.85% NaCl and confirmed using an API 20E kit. ISO 21872–1:2017 Method. —For the ISO 21872–1:2017 reference method, a 25 g test portion was homogenized in 225 mL of ASPW and incubated for 6 ± 1 h at 41.5 ± 1.0°C. After 6 h, 1 mL of the enriched sample was transferred into a tube containing 10 mL of ASPW and incubated at 41.5 ± 1.0°C for 18 ± 1 h. After incubation, 1 μL of sample was streaked onto TCBS and mCPC or CC agar. The TCBS plates were incubated at 37.0 ± 1.0°C for 24 ± 1 h, and the mCPC or CC agar was incubated at 39–40°C for 24 ± 1 h. After incubation, the plates were examined for typical colony morphology of the target strains ( see “BAM Chapter 9 Method” for colony morphology descriptions). A minimum of two suspect colonies were picked for confirmation and streaked onto Tryptone Soy Agar with 2% NaCl and incubated at 35.0 ± 2.0°C for 18–24 h. After incubation, one colony per sample was suspended in 0.85% NaCl and confirmed using an API 20E kit. The Thermo Scientific SureTect Vibrio cholerae, V. parahaemolyticus, and V. vulnificus PCR Assay. —All test portions were analyzed by following the SureTect Vibrio PCR Assay workflow after 8–20 h of enrichment for salmon roll with cream cheese and 16 h ± 10 min of enrichment for the remaining matrixes. All test portions were analyzed using the Applied Biosystems QuantStudio 5 Real-Time PCR Instrument and 7500 Fast Real-Time PCR Instrument. All portions, regardless of presumptive results, were culturally confirmed according to the BAM Chapter 9 reference method for all matrixes and the ISO 21872–1:2017 reference method for raw tuna and raw mussels. In addition, a candidate confirmation was performed as previously described in the section “Confirmation.” Most Probable Number Analysis. —For the BAM Chapter 9 method, the concentration of V. cholerae, V. parahaemolyticus, and V. vulnificus in the low-level inoculum for all 25 g test portions was determined by Most Probable Number (MPN) on the day of analysis by evaluating 20 × 25 g (BAM Ch. 9 reference method test portions), 5 × 50 g, and 5 × 10 g uninoculated portions. The concentration for each strain in the high-level inoculum was determined by MPN by evaluating 5 × 25 g (BAM Ch. 9 reference method test portions), 5 × 10 g, and 5 × 5 g inoculated test portions. For the 50 g portions 450 mL of APW was added, for the 10 g portions 90 mL of APW was used, and for the 5 g portions 45 mL of APW was used. For the ISO 21872–1:2017 reference method, the concentration of each target strain in the low-level inoculum for all test portions was determined by MPN on the day of analysis by evaluating 20 × 25 g (reference method test portions), 5 × 50 g, and 5 × 10 g uninoculated portions. For the high inoculum level, the concentration of each strain was determined by MPN by evaluating 5 × 25 g (refence method test portions), 5 × 10 g, and 5 × 5 g inoculated portions. For the 50 g portions 450 mL of APW was added, for the 10 g portions 90 mL of APW was used, and for the 5 g portions 45 mL of APW was used. The number of positives from the three test levels was used to calculate the MPN using the Least Cost Formulation Most Probable Number calculator, version 1.6 (Virginia Beach, VA) provided by AOAC RI. Results. —As per criteria outlined in Appendix J, fractional positive results were obtained for 50 g test portions of raw tuna, raw mussels, salmon roll with cream cheese, green-lipped mussel extract, and cooked shrimp for the SureTect Vibrio PCR Assay method. Prior to inoculation, an APC result of 3.9 × 10 2 cfu/g was established for cooked shrimp, 2.0 × 10 1 cfu/g for raw mussels, 9.1 × 10 3 cfu/g for salmon roll with cream cheese, 8.8 × 10 3 cfu/g for raw tuna, and 0 cfu/g for green lipped mussel extract. For the high spike level for the candidate and both reference methods, the number of positives detected was slightly below AOAC and ISO requirements. During preparation of inoculating culture, numerous attempts were made to achieve a higher spiking level, but unfortunately the number of positives detected at the higher level fell below the stipulated requirements. The candidate method detected 3/5 positives or above for all matrixes tested with a POD c of 0.60 or higher, compared to the reference methods, which detected anywhere from 0 to 5/5 positives for the high level. This is likely due to stress caused by the required fridge hold/heat-stress/desiccation (depending on matrix) required prior to analysis. Vibrio spp. are well documented for undergoing genotypic and, consequently, phenotypic changes when under stress (particularly cold stress) to ensure survival through alteration of different metabolic pathways, which then leads to a lag in growth phase when transferred to normal growth conditions and typical pathways must be upregulated again ( ). A study looking at temperature upshift in V. parahaemolyticus after cold stressed found it took up to almost 48 h for cells to return to typical growth (normal cellular shape and upregulation of certain metabolic pathways, for example) ( ). This is likely why the Vibrio strains had recovered to a fractional level at the time of analysis but did not meet the higher spike level target. Evidence for this can also be seen when looking at the individual matrixes. For the processed GLME and cooked shrimp matrixes, the candidate method had identical performance to the BAM reference method, and both methods hit the stipulated spike level of 5/5 positives. This is likely because the incubation timepoints were more comparable 18–24 h for BAM and 16 h for candidate (BAM method stipulates reincubation for processed matrixes, as mentioned in the previous section) between the reference methods and longer compared to the unprocessed matrixes, which allowed a longer time for stressed cell recovery. However, for the non-processed matrixes (raw tuna, raw mussels, and salmon roll with cream cheese), where the reference method timepoints were substantially shortened (6–8 h for BAM compared to 18–24 h for the processed matrixes), there was a notable difference in high spike positives detected. The candidate method detected 3/5 positives at the high level for all matrixes compared to 0/5, 2/5, and 1/5 for the BAM method, respectively. The candidate method timepoints were 16 h for both raw tuna and raw mussels, and for salmon roll with cream cheese it was 8 h; however, prewarmed media is used to support cell recovery. For the ISO reference method, even though the timepoint was 18–24 h, there is the added complication of higher background flora associated with raw tuna ( ), which can cause difficulties in recovering cells due to competition between species. The ISO data were more comparable to the candidate data, however, 2/5 vs 3/5 positives, respectively, likely due to the slightly longer timepoint. Overall, this demonstrates that the shorter incubation did not allow enough time for the Vibrio to recover to the levels anticipated in a higher spike. Furthermore, the performance of the matrixes would suggest that the genotypic and phenotypic changes seen within Vibrio in stress-inducing situations appear to be more severe in cold environments compared to heat and desiccation, as recovery in GLME and cooked shrimp was far more substantial than the matrixes that underwent a fridge hold. For the candidate method, the fractional spike levels were hit for all matrixes, and while the high level did not hit 5/5 positives for every matrix, as there were no issues with detection of a lower spike level this lends confidence to the ability of the assay to detect higher cell loads; therefore, the lower positives achieved here should not warrant concern for performance of the assay. The POD was calculated as the number of positive outcomes divided by the total number of trials using the Least Cost Formulations, Ltd, AOAC Binary Data Interlaboratory Study Workbook version 5.2 (Virgina Beach, VA) ( ). The POD was calculated for the candidate presumptive results, POD CP , the candidate confirmatory results, POD CC , the difference in the candidate presumptive and confirmatory results, dPOD CP, presumptive candidate results that confirmed positive, POD C, the reference method, POD R , and the difference in the confirmed candidate and reference methods, dPOD C . The POD analysis between the SureTect Vibrio PCR Assay and reference methods indicated that there was no significant difference at the 5% confidence level between the number of positive results by the methods for raw mussels, green-lipped mussel extract, and cooked shrimp matrixes for the BAM method and the raw tuna and raw mussel matrixes for the ISO method. For raw tuna and the salmon roll with cream cheese, POD analysis showed superior performance of the candidate method due to poor performance of the BAM reference method. For raw tuna, the SureTect Vibrio PCR Assay detected eight confirmed positives for the low level compared to zero positives detected by the BAM method; this was also the case for the high level, with three positives confirmed from the SureTect Vibrio PCR Assay compared to zero from the BAM method. For the salmon roll with cream cheese, the SureTect Vibrio PCR Assay detected nine confirmed positives for the low level compared to two for the BAM method. For the high level, although not statistically significant, the SureTect Vibrio PCR Assay detected three confirmed positives compared to just one with the BAM method. In general, although not statistically significant for all matrixes, the data show a trend of higher detection for the SureTect Vibrio PCR Assay compared to the BAM method. The POD analysis between the SureTect Vibrio PCR Assay method presumptive and confirmed results indicated no significant difference at the 5% level for all methods following both reference confirmation method procedures and the candidate method confirmation procedure except for raw tuna, in which 18 presumptive positives were detected compared to eight confirmed using the reference confirmation. A summary of the POD analyses is presented in – . Salmon roll with cream cheese: Further investigative study and results. —The SureTect Vibrio PCR assay is a multiplex PCR assay and therefore can be used to detect all three target strains of V. cholerae, V. parahaemolyticus, and V. vulnificus from one single sample whether one, two, or all three target strains are present. As such, the initial study design was to triple-infect the salmon roll with cream cheese as outlined previously to demonstrate the multiplex capability of the assay. However, it is well documented that multiple Vibrio spp. exhibit both inter- and intra-specific competition to occupy and thrive within the same ecological niche ( ). For example, both V. parahaemolyticus and V. cholerae use the well-characterized type VI secretory system to compete with other bacterial species, other Vibrio spp., and different strains of V. cholerae and V. parahaemolyticus by secreting anti-eukaryotic and prokaryotic effectors ( , ). The geographical spread (origin of the sample) and seasonality of each target strain also need to be considered. For example, populations of V. parahaemolyticus in the Pacific Northwest of the US peak in June but then wane throughout the rest of the year ( ). This contrasts with the coast of South Korea, where V. parahaemolyticus remains ubiquitous all year round compared to V. cholerae and V. vulnificus , which are seasonally affected and isolated specifically during the summer months ( ). The inherent competition, geographical spread, and seasonality between the different strains mean it is unlikely to see a sample that is dual or triple infected, but not impossible. If this is the case, further difficulty is encountered when trying to isolate and confirm suspect colonies on culture media due to the differing growth rates between the target strains. During the salmon roll with cream cheese matrix study, there was PCR detection and amplification of the three spiked strains, but considerable difficulty in confirming these on agar. Looking at the generated PCR C t values showed a 1–2 log difference in growth of the target strains as anticipated due to the differing growth rates between targets. Therefore, this 1–2 log difference had likely caused overgrowth of the higher-density strain on the plate, which negated confirmation of the other species present. A multiplex study was devised to demonstrate the capability of the SureTect Vibrio PCR Assay method to detect all three target strains and to also demonstrate the difficulties encountered in isolation and confirmation of all three target species when present at different growth levels. The study looked at several combinations of all three target organisms with a 1–2 log difference compared to a combination where each target was spiked at the same level, and an uninoculated/screening level in salmon roll with cream cheese. All combinations were spiked post-enrichment to negate the effect of the different growth rates and run on the QuantStudio 5 PCR Instrument; all combinations were then plated onto TCBS, CHROMagar Vibrio, and mCPC agar to assess recovery. All combinations are outlined in , and agar scoring is outlined in . The results of the study were as expected. When the difference between the strains was 2-log growth, recovery of all three target strains was not possible on agar despite positive detection by PCR. When the difference was 1-log, it was possible to isolate two of the three target strains, and when all three target strains were at the same growth level, all three were recoverable on agar. The decision was made to use only the V. parahaemolyticus data in this matrix and not to continue with triple inoculation. The results of the multiplex spike study are outlined in and and . Robustness study For the robustness study, the SureTect Vibrio PCR Assay method was evaluated by adjusting three testing parameters. The three parameters included: enrichment time (7, 8, and 24 h), volume of sample lysed (9, 10, and 11 μL), and volume of lysate (18, 20, and 22 μL). A total of nine combinations were evaluated, with the ninth combination prepared as nominal conditions to provide a comparison. Methodology. —Twenty 50 g test portions of salmon roll with cream cheese were analyzed. Ten were inoculated with V. cholerae at a level to yield fractional positive results, and 10 were uninoculated. All 50 g test portions of salmon roll with cream cheese were enriched in 450 ± 10 mL of ASPW and incubated according to the enrichment times summarized in . All portions were randomized and blind-coded. Results. —POD analysis of the performance of the SureTect Vibrio PCR assay method demonstrated no statistically significant differences between the nominal conditions and changes in testing parameters. However, the shorter enrichment time of 7 h using the 7500 Fast or the QuantStudio 5 PCR Instrument impacted the number of positives detected by the SureTect Vibrio Assay. Although the POD analysis showed no statistically significant difference between the nominal conditions and the shorter timepoint as mentioned above, the number of positives detected at 7 and 8 h (1 versus 4/5, respectively, depending on instrument) suggests that additional time for incubation is necessary. Therefore, the assay is robust at the longer incubation timepoint but not at the shorter timepoint, indicating that the sample must be incubated for the minimum time specified. The POD results and 95% confidence intervals for each target analyte and treatment combination are presented in and . 

The matrix study was conducted according to the AOAC guidelines Appendix J following an unpaired study design for 50 g of raw tuna, 50 g of raw mussels, 50 g of salmon roll with cream cheese, 50 g of green-lipped mussel extract, and 125 g of cooked shrimp. The SureTect Vibrio PCR Assay method was compared to the qualitative version of the BAM Chapter 9 reference method for all matrixes and additionally to the ISO 21872–1:2017 reference method for raw tuna and raw mussels. The BAM Chapter 9 reference method followed the detection principle as indicated for V. cholerae as opposed to the MPN procedure for V. parahaemolyticus and V. vulnificus to allow for comparison to the candidate nonquantitative detection method. Since MPN methods are based on a series of qualitative methods, it was determined that using the qualitative method found in the BAM MPN to assess performance of the candidate method was appropriate. The method used the same enrichment as detailed in BAM Chapter 9, and the sample enrichment ratio was the same as outlined in BAM Chapter 1, section G.1 ( ). This allowed for direct comparison of the candidate qualitative method to the FDA reference methodology for detection while adhering to determined enrichment procedures and ratios. In addition, a candidate confirmation procedure was included in the study as previously described in the “Confirmation” section. Methodology. —Raw tuna, raw mussels, salmon roll with cream cheese, green-lipped mussel extract, and cooked shrimp were purchased from a local supplier and prescreened for natural contamination of V. cholerae, V. parahaemolyticus, and V. vulnificus following the candidate method. The mesophilic aerobic plate count (APC) was determined following the BAM Chapter 3 Aerobic Plate Count reference method ( ). No natural contamination was detected in the screening of all five products; however, late amplification was seen in the cooked shrimp matrix and one batch of the green-lipped mussel extract, indicating potential low-level contamination or the presence of dead cells. The presence of dead cells rather than viable low-level contamination was determined by nonselective and selective plating of the enrichment, as well as by screening of the matrix at 0 and 16 h and a comparison of PCR cycle threshold (C t ) values. The C t value did not change between the timepoints. Had there been low-level contamination present, the C t value would have decreased (i.e., 36 to 27 due to growth of the contaminant during incubation). However, as the C t value did not change and growth was not recoverable on any agar plate, the PCR amplification seen during screening was most likely due to dead cell DNA. This issue was addressed by performing a 1-in-10 dilution after enrichment that removed the dead cell amplification (all samples had very late C t values—greater than 35), as described in the troubleshooting steps in the instructions for use should an end user encounter this problem. The matrix study consisted of evaluating a total of thirty 50 g portions of raw tuna, raw mussels, salmon roll with cream cheese, and green-lipped mussel extract and 125 g portions of cooked shrimp following an unpaired study design. Within each sample set, there were five non-inoculated portions (POD = 0), 20 low-level inoculated portions (targeting POD = 0.25–0.75), and five high-level inoculated portions (targeting POD = 1). For the raw tuna, raw mussels, and salmon roll with cream cheese, inoculation was conducted with a liquid culture. Raw tuna was inoculated with V. cholerae , raw mussels were inoculated with V. parahaemolyticus , and salmon roll with cream cheese was inoculated with all three target species. These matrixes were artificially contaminated as follows. For the raw tuna, V. cholerae was grown in ASPW for 24 h at 35 ± 1°C, and then serially diluted with Dulbecco’s Phosphate Buffered Saline (DPBS) and plated onto TSA with 2% NaCl. The culture level was determined and the amount of inoculum to add to the matrix was calculated based on the target level. The raw tuna was cut, placed into a large sterile bag, and homogenized by hand-mixing. Low- and high-level contaminated tuna was prepared by weighing the appropriate amount of tuna into a bag and inoculating with a dilution of V. cholerae to obtain fractional recovery for the low level and all positives for the high level. The bags of inoculated raw tuna were stored at 2–8°C for 72 h. After storage, 25 g of inoculated tuna was combined with 25 g of non-inoculated tuna to create the 50 g test portion on the day of analysis. For the raw mussels, V . parahaemolyticus was grown in ASPW for 24 h at 35 ± 1°C, and then serially diluted with DPBS and plated onto TSA with 2% NaCl. The culture level was determined and the amount of inoculum to add to the matrix was calculated based on the target level. The raw mussels were removed from the shell, cut up, placed in a large sterile bag, and homogenized by hand-mixing. Low- and high-level contaminated mussels were prepared by weighing the appropriate amount into a bag and inoculating with a dilution of V. parahaemolyticus to obtain fractional recovery in the low level and all positives in the high level. The bags of inoculated raw mussels were stored at 2–8°C for 72 h. After storage, a 25 g portion was removed and combined with 25 g of non-inoculated mussels to create the 50 g test portion on the day of analysis. For the salmon roll with cream cheese, each target strain ( V. cholerae, V . parahaemolyticus, and V. vulnificus ) was grown in ASPW for 24 h at 35 ± 1°C and then serially diluted with DPBS and plated onto TSA with 2% NaCl. The culture level was determined and the amount of inoculum to add to the matrix was calculated based on the target level. The salmon roll was cut up, transferred to a large sterile bag and homogenized with cream cheese by hand mixing; the bag contained 70% salmon and 30% cream cheese. Low- and high-level contaminated materials were prepared by weighing the appropriate amount of homogenized salmon roll into a bag and inoculating with a dilution of each strain to obtain fractional recovery in the low level and all positives in the high level. The bags of inoculated salmon roll with cream cheese were stored at 2–8°C for 72 h. After storage, a 25 g portion was removed and combined with 25 g of non-inoculated material to create the 50 g test portion on the day of analysis. For the green-lipped mussel extract, inoculation was conducted with a lyophilized culture of V. parahaemolyticus . The culture was grown in 400 mL of ASPW for 24 h at 35.0 ± 1.0°C and pelleted by centrifugation. Four grams of gelatin, 5 g NaCl, and 25 g skimmed milk were mixed in 225 mL of phosphate buffer, 10 mL of which was then added to the spun-down culture pellet and vortexed until the pellet fully dissolved. The 10 mL of dissolved pellet was transferred to a glass flask and then placed in a −80°C freezer for 30 min. After 30 min, the glass flask was placed in a freeze-dryer for 24 h. After 24 h, the glass flask was removed, and the dried culture was crushed into a fine powder. The fine powder was added to 200 g of green-lipped mussel extract to make the seed culture. The seed remained at room temperature (18–25°C) for 48 h before being diluted with uninoculated green-lipped mussel extract. Typically, room temperature stable products must equilibrate for 2 weeks before testing, but because the Vibrio inoculum would not survive that long, AOAC agreed that the equilibration time could be reduced to 48 h. High and low contamination levels were prepared using the diluted seed culture and homogenized. After storage, a 25 g portion was removed and combined with 25 g of non-inoculated material to create the 50 g test portion on the day of analysis. For the cooked shrimp, a liquid heat-stressed culture of V. vulnificus was used. The culture was grown in ASPW for 48 h at 35 ± 1°C and then serially diluted in DPBS. The culture was heat-stressed at 46°C for 50 s, and then plated onto TSA with and without 2% NaCl. Percentage of injury was determined by counting the number of colonies on the selective agar, dividing by the number of colonies on the nonselective agar, subtracting from 1, and then multiplying by 100 (50–80% injury was required). The inoculating culture was found to have an injury level of 77%. Shrimp were cut up, transferred to a large sterile bag using sterile scissors, and then hand-homogenized. A low-level and high-level bag of cooked shrimp was weighed and then inoculated with a specific target level. The inoculated cooked shrimp were then stored at 4°C for 48 h. After storage, a 25 g portion was removed and combined with 100 g of non-inoculated material to create the 125 g test portion on the day of analysis. BAM Chapter 9 Method. —The BAM Chapter 9 reference method was followed as described for V. cholerae , in agreement with AOAC to detect all Vibrio species. The 25 g test portions were weighed into a tared jar and enriched with 225 mL of APW and then mixed thoroughly for 2 min. Test portions were incubated for 6–8 h at 35 ± 2°C and reincubated overnight if the sample had been processed (cooked shrimp, green-lipped mussel extract). After incubation, a 3 mm loopful of the surface pellicle of the APW culture was streaked onto TCBS and mCPC or CC agar plates. The TCBS plates were incubated for 18–24 h at 35 ± 2°C, and the mCPC or CC plates were incubated for 18–24 h at 39–40°C. Following incubation, the plates were examined for typical colony morphology of the target organism. Typical colonies of V. cholerae on TCBS agar are large (2–3 mm), smooth, yellow, and slightly flattened with opaque centers and translucent peripheries. Typical colonies of V. cholerae on mCPC or CC agar are small, smooth, opaque, and green to purple in color, with a purple background on extended incubation. Typical colonies of V. parahaemolyticus on TCBS agar are round, opaque, green, or bluish. Most strains of V. parahaemolyticus will not grow on mCPC or CC agar. If growth occurs, colonies will be green-purple in color due to lack of cellobiose fermentation. Typical colonies of V. vulnificus on TCBS agar are round, opaque, green, or bluish, and typical colonies of V. vulnificus on mCPC or CC agar are round, flat, opaque, yellow, and 1–2 mm in diameter. A minimum of two suspect colonies were picked for confirmation and streaked onto TSA with 2% NaCl and incubated at 35 ± 2°C for 18–24 h. After incubation, one colony per sample was suspended in 0.85% NaCl and confirmed using an API 20E kit. ISO 21872–1:2017 Method. —For the ISO 21872–1:2017 reference method, a 25 g test portion was homogenized in 225 mL of ASPW and incubated for 6 ± 1 h at 41.5 ± 1.0°C. After 6 h, 1 mL of the enriched sample was transferred into a tube containing 10 mL of ASPW and incubated at 41.5 ± 1.0°C for 18 ± 1 h. After incubation, 1 μL of sample was streaked onto TCBS and mCPC or CC agar. The TCBS plates were incubated at 37.0 ± 1.0°C for 24 ± 1 h, and the mCPC or CC agar was incubated at 39–40°C for 24 ± 1 h. After incubation, the plates were examined for typical colony morphology of the target strains ( see “BAM Chapter 9 Method” for colony morphology descriptions). A minimum of two suspect colonies were picked for confirmation and streaked onto Tryptone Soy Agar with 2% NaCl and incubated at 35.0 ± 2.0°C for 18–24 h. After incubation, one colony per sample was suspended in 0.85% NaCl and confirmed using an API 20E kit. The Thermo Scientific SureTect Vibrio cholerae, V. parahaemolyticus, and V. vulnificus PCR Assay. —All test portions were analyzed by following the SureTect Vibrio PCR Assay workflow after 8–20 h of enrichment for salmon roll with cream cheese and 16 h ± 10 min of enrichment for the remaining matrixes. All test portions were analyzed using the Applied Biosystems QuantStudio 5 Real-Time PCR Instrument and 7500 Fast Real-Time PCR Instrument. All portions, regardless of presumptive results, were culturally confirmed according to the BAM Chapter 9 reference method for all matrixes and the ISO 21872–1:2017 reference method for raw tuna and raw mussels. In addition, a candidate confirmation was performed as previously described in the section “Confirmation.” Most Probable Number Analysis. —For the BAM Chapter 9 method, the concentration of V. cholerae, V. parahaemolyticus, and V. vulnificus in the low-level inoculum for all 25 g test portions was determined by Most Probable Number (MPN) on the day of analysis by evaluating 20 × 25 g (BAM Ch. 9 reference method test portions), 5 × 50 g, and 5 × 10 g uninoculated portions. The concentration for each strain in the high-level inoculum was determined by MPN by evaluating 5 × 25 g (BAM Ch. 9 reference method test portions), 5 × 10 g, and 5 × 5 g inoculated test portions. For the 50 g portions 450 mL of APW was added, for the 10 g portions 90 mL of APW was used, and for the 5 g portions 45 mL of APW was used. For the ISO 21872–1:2017 reference method, the concentration of each target strain in the low-level inoculum for all test portions was determined by MPN on the day of analysis by evaluating 20 × 25 g (reference method test portions), 5 × 50 g, and 5 × 10 g uninoculated portions. For the high inoculum level, the concentration of each strain was determined by MPN by evaluating 5 × 25 g (refence method test portions), 5 × 10 g, and 5 × 5 g inoculated portions. For the 50 g portions 450 mL of APW was added, for the 10 g portions 90 mL of APW was used, and for the 5 g portions 45 mL of APW was used. The number of positives from the three test levels was used to calculate the MPN using the Least Cost Formulation Most Probable Number calculator, version 1.6 (Virginia Beach, VA) provided by AOAC RI. Results. —As per criteria outlined in Appendix J, fractional positive results were obtained for 50 g test portions of raw tuna, raw mussels, salmon roll with cream cheese, green-lipped mussel extract, and cooked shrimp for the SureTect Vibrio PCR Assay method. Prior to inoculation, an APC result of 3.9 × 10 2 cfu/g was established for cooked shrimp, 2.0 × 10 1 cfu/g for raw mussels, 9.1 × 10 3 cfu/g for salmon roll with cream cheese, 8.8 × 10 3 cfu/g for raw tuna, and 0 cfu/g for green lipped mussel extract. For the high spike level for the candidate and both reference methods, the number of positives detected was slightly below AOAC and ISO requirements. During preparation of inoculating culture, numerous attempts were made to achieve a higher spiking level, but unfortunately the number of positives detected at the higher level fell below the stipulated requirements. The candidate method detected 3/5 positives or above for all matrixes tested with a POD c of 0.60 or higher, compared to the reference methods, which detected anywhere from 0 to 5/5 positives for the high level. This is likely due to stress caused by the required fridge hold/heat-stress/desiccation (depending on matrix) required prior to analysis. Vibrio spp. are well documented for undergoing genotypic and, consequently, phenotypic changes when under stress (particularly cold stress) to ensure survival through alteration of different metabolic pathways, which then leads to a lag in growth phase when transferred to normal growth conditions and typical pathways must be upregulated again ( ). A study looking at temperature upshift in V. parahaemolyticus after cold stressed found it took up to almost 48 h for cells to return to typical growth (normal cellular shape and upregulation of certain metabolic pathways, for example) ( ). This is likely why the Vibrio strains had recovered to a fractional level at the time of analysis but did not meet the higher spike level target. Evidence for this can also be seen when looking at the individual matrixes. For the processed GLME and cooked shrimp matrixes, the candidate method had identical performance to the BAM reference method, and both methods hit the stipulated spike level of 5/5 positives. This is likely because the incubation timepoints were more comparable 18–24 h for BAM and 16 h for candidate (BAM method stipulates reincubation for processed matrixes, as mentioned in the previous section) between the reference methods and longer compared to the unprocessed matrixes, which allowed a longer time for stressed cell recovery. However, for the non-processed matrixes (raw tuna, raw mussels, and salmon roll with cream cheese), where the reference method timepoints were substantially shortened (6–8 h for BAM compared to 18–24 h for the processed matrixes), there was a notable difference in high spike positives detected. The candidate method detected 3/5 positives at the high level for all matrixes compared to 0/5, 2/5, and 1/5 for the BAM method, respectively. The candidate method timepoints were 16 h for both raw tuna and raw mussels, and for salmon roll with cream cheese it was 8 h; however, prewarmed media is used to support cell recovery. For the ISO reference method, even though the timepoint was 18–24 h, there is the added complication of higher background flora associated with raw tuna ( ), which can cause difficulties in recovering cells due to competition between species. The ISO data were more comparable to the candidate data, however, 2/5 vs 3/5 positives, respectively, likely due to the slightly longer timepoint. Overall, this demonstrates that the shorter incubation did not allow enough time for the Vibrio to recover to the levels anticipated in a higher spike. Furthermore, the performance of the matrixes would suggest that the genotypic and phenotypic changes seen within Vibrio in stress-inducing situations appear to be more severe in cold environments compared to heat and desiccation, as recovery in GLME and cooked shrimp was far more substantial than the matrixes that underwent a fridge hold. For the candidate method, the fractional spike levels were hit for all matrixes, and while the high level did not hit 5/5 positives for every matrix, as there were no issues with detection of a lower spike level this lends confidence to the ability of the assay to detect higher cell loads; therefore, the lower positives achieved here should not warrant concern for performance of the assay. The POD was calculated as the number of positive outcomes divided by the total number of trials using the Least Cost Formulations, Ltd, AOAC Binary Data Interlaboratory Study Workbook version 5.2 (Virgina Beach, VA) ( ). The POD was calculated for the candidate presumptive results, POD CP , the candidate confirmatory results, POD CC , the difference in the candidate presumptive and confirmatory results, dPOD CP, presumptive candidate results that confirmed positive, POD C, the reference method, POD R , and the difference in the confirmed candidate and reference methods, dPOD C . The POD analysis between the SureTect Vibrio PCR Assay and reference methods indicated that there was no significant difference at the 5% confidence level between the number of positive results by the methods for raw mussels, green-lipped mussel extract, and cooked shrimp matrixes for the BAM method and the raw tuna and raw mussel matrixes for the ISO method. For raw tuna and the salmon roll with cream cheese, POD analysis showed superior performance of the candidate method due to poor performance of the BAM reference method. For raw tuna, the SureTect Vibrio PCR Assay detected eight confirmed positives for the low level compared to zero positives detected by the BAM method; this was also the case for the high level, with three positives confirmed from the SureTect Vibrio PCR Assay compared to zero from the BAM method. For the salmon roll with cream cheese, the SureTect Vibrio PCR Assay detected nine confirmed positives for the low level compared to two for the BAM method. For the high level, although not statistically significant, the SureTect Vibrio PCR Assay detected three confirmed positives compared to just one with the BAM method. In general, although not statistically significant for all matrixes, the data show a trend of higher detection for the SureTect Vibrio PCR Assay compared to the BAM method. The POD analysis between the SureTect Vibrio PCR Assay method presumptive and confirmed results indicated no significant difference at the 5% level for all methods following both reference confirmation method procedures and the candidate method confirmation procedure except for raw tuna, in which 18 presumptive positives were detected compared to eight confirmed using the reference confirmation. A summary of the POD analyses is presented in – . Salmon roll with cream cheese: Further investigative study and results. —The SureTect Vibrio PCR assay is a multiplex PCR assay and therefore can be used to detect all three target strains of V. cholerae, V. parahaemolyticus, and V. vulnificus from one single sample whether one, two, or all three target strains are present. As such, the initial study design was to triple-infect the salmon roll with cream cheese as outlined previously to demonstrate the multiplex capability of the assay. However, it is well documented that multiple Vibrio spp. exhibit both inter- and intra-specific competition to occupy and thrive within the same ecological niche ( ). For example, both V. parahaemolyticus and V. cholerae use the well-characterized type VI secretory system to compete with other bacterial species, other Vibrio spp., and different strains of V. cholerae and V. parahaemolyticus by secreting anti-eukaryotic and prokaryotic effectors ( , ). The geographical spread (origin of the sample) and seasonality of each target strain also need to be considered. For example, populations of V. parahaemolyticus in the Pacific Northwest of the US peak in June but then wane throughout the rest of the year ( ). This contrasts with the coast of South Korea, where V. parahaemolyticus remains ubiquitous all year round compared to V. cholerae and V. vulnificus , which are seasonally affected and isolated specifically during the summer months ( ). The inherent competition, geographical spread, and seasonality between the different strains mean it is unlikely to see a sample that is dual or triple infected, but not impossible. If this is the case, further difficulty is encountered when trying to isolate and confirm suspect colonies on culture media due to the differing growth rates between the target strains. During the salmon roll with cream cheese matrix study, there was PCR detection and amplification of the three spiked strains, but considerable difficulty in confirming these on agar. Looking at the generated PCR C t values showed a 1–2 log difference in growth of the target strains as anticipated due to the differing growth rates between targets. Therefore, this 1–2 log difference had likely caused overgrowth of the higher-density strain on the plate, which negated confirmation of the other species present. A multiplex study was devised to demonstrate the capability of the SureTect Vibrio PCR Assay method to detect all three target strains and to also demonstrate the difficulties encountered in isolation and confirmation of all three target species when present at different growth levels. The study looked at several combinations of all three target organisms with a 1–2 log difference compared to a combination where each target was spiked at the same level, and an uninoculated/screening level in salmon roll with cream cheese. All combinations were spiked post-enrichment to negate the effect of the different growth rates and run on the QuantStudio 5 PCR Instrument; all combinations were then plated onto TCBS, CHROMagar Vibrio, and mCPC agar to assess recovery. All combinations are outlined in , and agar scoring is outlined in . The results of the study were as expected. When the difference between the strains was 2-log growth, recovery of all three target strains was not possible on agar despite positive detection by PCR. When the difference was 1-log, it was possible to isolate two of the three target strains, and when all three target strains were at the same growth level, all three were recoverable on agar. The decision was made to use only the V. parahaemolyticus data in this matrix and not to continue with triple inoculation. The results of the multiplex spike study are outlined in and and . 

For the robustness study, the SureTect Vibrio PCR Assay method was evaluated by adjusting three testing parameters. The three parameters included: enrichment time (7, 8, and 24 h), volume of sample lysed (9, 10, and 11 μL), and volume of lysate (18, 20, and 22 μL). A total of nine combinations were evaluated, with the ninth combination prepared as nominal conditions to provide a comparison. Methodology. —Twenty 50 g test portions of salmon roll with cream cheese were analyzed. Ten were inoculated with V. cholerae at a level to yield fractional positive results, and 10 were uninoculated. All 50 g test portions of salmon roll with cream cheese were enriched in 450 ± 10 mL of ASPW and incubated according to the enrichment times summarized in . All portions were randomized and blind-coded. Results. —POD analysis of the performance of the SureTect Vibrio PCR assay method demonstrated no statistically significant differences between the nominal conditions and changes in testing parameters. However, the shorter enrichment time of 7 h using the 7500 Fast or the QuantStudio 5 PCR Instrument impacted the number of positives detected by the SureTect Vibrio Assay. Although the POD analysis showed no statistically significant difference between the nominal conditions and the shorter timepoint as mentioned above, the number of positives detected at 7 and 8 h (1 versus 4/5, respectively, depending on instrument) suggests that additional time for incubation is necessary. Therefore, the assay is robust at the longer incubation timepoint but not at the shorter timepoint, indicating that the sample must be incubated for the minimum time specified. The POD results and 95% confidence intervals for each target analyte and treatment combination are presented in and . 

The SureTect Vibrio PCR assay method successfully detected all spiked target organisms in 50 g of raw tuna, 50 g of raw mussels, 50 g of salmon roll with cream cheese, 50 g of green-lipped mussel extract, and 125 g of cooked shrimp. The candidate method tested 50 g portions compared to 25 g portions for the ISO and BAM (detection principle) reference method. POD analysis of the data showed no statistically significant differences between the candidate method and the ISO reference method but did demonstrate significant differences in favor of the candidate method when compared to the BAM method for raw tuna and salmon roll with cream cheese. Furthermore, the matrix data demonstrated the high sensitivity and robust performance of the candidate method considering the differing test portion sizes but uniform spiking levels. POD analysis also showed no statistically significant differences between presumptive positives and confirmed positives for the candidate method for any of the matrixes, except raw tuna in which more presumptive positives than confirmed positives were produced (18 presumptive positive results with 8 confirmed). The challenge with the raw tuna matrix was likely due to the high level of background flora present. It is well documented that raw tuna has a high level of background flora ( ), which greatly complicates the isolation of suspect colonies, due to overgrowth of non-target organisms. The APC count for raw tuna was high and similar to that for salmon roll with cream cheese, where culture confirmation was also difficult following the initial study design. The salmon roll with cream cheese was triple-inoculated with all three target organisms, and only V. parahaemolyticus was culturally confirmed. Vibrio parahaemolyticus has a faster growth rate compared to both V. cholerae (which was the spike organism in raw tuna) and V. vulnificus . As mentioned in the multiplex spike study section above, Vibrio spp. are well documented for secreting extracellular protein effectors in competition with both other Vibrio spp. and other background organisms that may be present ( , ). The salmon roll with cream cheese was spiked with three Vibrio targets compared to raw tuna, meaning there was a higher level of effector proteins likely combating growth of background flora. This, in combination with the faster growth rate of V. parahaemolyticus compared to V. cholerae, is likely why confirmation of V. parahaemolyticus in the salmon roll with cream cheese was not overly challenging compared to confirming the V. cholerae in raw tuna. There was also comparable confirmation performance to the ISO reference method in which only seven positives were confirmed; the BAM method failed to detect and confirm any positives. Therefore, the SureTect Vibrio PCR Assay method is a highly sensitive method able to detect potential samples at risk when culture confirmation fails due to interference from high levels of background flora that the PCR technology negates. For cooked shrimp, no statistically significant differences were seen between the candidate versus reference and presumptive versus confirmed results. However, there was an important technical challenge encountered during the study that must be noted. The cooked shrimp matrix routinely returned PCR positives for the unspiked targets (the matrix was spiked with V. vulnificus and routinely returned V. parahaemolyticus and V. cholerae positive results). Initial thoughts were that natural contamination had occurred, but this was deemed unlikely in this cooked, ready-to-eat matrix. In addition, culturing of the samples also failed to return any suspect V. parahaemolyticus or V. cholerae colonies. Review of the PCR amplification plots showed that the non-spiked organisms’ amplification typically had very late C t values (33–40). Coupled with the failure to isolate these strains on agar after enrichment led to the conclusion that these amplifications were likely from dead cell DNA. Vibrio spp. are typically ubiquitous within shrimp ( ), and, prior to cooking, levels high enough for detection are likely to be present. Given that Vibrio spp. are Gram-negative, the cooking/boiling process easily denatures the bacterial cell wall, allowing free-floating DNA to be released into the matrix. In addition, if dead cells are present but DNA has not been released, the lysis procedure of the SureTect Vibrio PCR Assay workflow will break down any remaining dead Vibrio cell walls to release the DNA. During PCR, this free-floating/dead cell DNA is amplified, leading to a registered positive by the software. To confirm the presence of dead cells rather than low-level contamination, as the workflow does not include a free DNA wash step, test portions were prepared and tested at 0, 16, and 24 h of enrichment to compare C t values. Portions were also plated at 0, 16, and 24 h to ensure that no suspect colonies were present. The C t values for all non-spiked amplifications were constant at all timepoints with no suspect growth seen on any of the culture media plates. Had the positive PCR call been due to low-level contamination, after 16 and 24 h of incubation the C t value would decrease to show an increase in the cell load present in the portion due to cell division during incubation, but as this did not occur, the call was due to the presence of dead cells. To negate this effect, a post-enrichment 1-in-10 dilution was added to the instructions for use that diluted the dead cell DNA to lower the C t value. This does not risk screening out low-level-contaminated samples since 16 h of incubation would typically result in a sufficiently high cell load to trigger a positive PCR result. The salmon roll investigation study demonstrated the natural competition that exists between different Vibrio strains and established the challenge of culture confirming dual- or triple-inoculated test portions. The results show that the kit is a consistent and capable multiplex assay and was able to easily detect the presence of all three spiked strains, whereas the culture confirmation struggled due to difference in growth rates, natural competition, and in a few cases breakthrough growth of background flora. The inclusivity and exclusivity studies correctly detected all 155 inclusivity isolates tested and excluded all 50 exclusivity isolates tested, highlighting the specificity of the method. The real-time stability study results and consequential POD analysis demonstrated no significant differences between kit lots, showing that manufacture and performance are equivalent between kit lots and demonstrating no overall degradation of the product over time, supporting the shelf-life statement. In the robustness study, no statistically significant differences were seen between the nominal and test conditions for the later enrichment timepoint, demonstrating that typical small parameter deviations that might occur when performed by an end user do not impact assay performance. For the 7 h timepoint, there were no statistically significant differences, but the POD confidence interval was very close to the limit for equivalence, with notably fewer positives at 7 h compared to the nominal conditions. This means that samples must be incubated for the minimum time specified. 

The data presented in this report, within their statistical uncertainty, support the product claims that the Thermo Scientific SureTect Vibrio cholerae, V. parahaemolyticus, and V. vulnificus PCR Assay provides a rapid and reliable means to detect V. cholerae , V. parahaemolyticus , and V. vulnificus in seafood matrixes. The results obtained by the POD analysis of the matrix study demonstrated either equivalent or superior performance of the candidate method to the reference methods for all matrixes tested. Apart from raw tuna, no statistically significant differences were seen between the presumptive and confirmed results for the candidate method. For raw tuna, the presumptive positive results were higher than the confirmed results, but it is commonly known that high background microflora can create issues in spotting characteristic colonies on selective agar plates. The inclusivity and exclusivity studies demonstrated 100% specificity of the method, and the product consistency and stability study demonstrated no difference in assay performance in kit lots at different expiration time points. The robustness study demonstrated that small deviations in testing parameters did not impact the performance of the assay at the later timepoint, but that performance was impacted at the shorter timepoint, meaning that samples must be incubated for the minimum time specified. In addition, no statistically significant differences were observed between the alternative confirmation and the traditional reference method confirmation. The SureTect Vibrio PCR Assay method is robust, quick, and simple to perform, providing results in approximately 80 min post-enrichment. The QuantStudio 5 and 7500 PCR Instrumentation provides flexibility, and the software is user-friendly and easy to use. 

