all available sequences of the targets of interest from databases such as genbank should be utilized to identify conserved areas which are suitable targets .
where a large number of targets are being amplified in a single mpcr assay , each amplified sequence should be of similar length , and should not exceed 300 base pairs to avoid competition .
an alternative application of the method is to amplify one longer target using primers against conserved regions and to use multiple probes to identify sequence variation within the amplicon . in this instance
the sigma aldrich dna calculator ( http://www.sigma-genosys.com/calc/dnacalc.asp ) can be used to reliably predict these features .
dna probes should be designed to have annealing temperatures close to 60c . to maximize specificity , two probes for each target of interest
can be included in the assay , one hybridizing to the forward dna strand adjacent to the reverse primer binding site , and the other to the reverse strand adjacent to the forward primer binding site . in this case , both forward and reverse primers must be biotin - modified . if only one probe per amplified target is being used , then only one primer need be biotin modified .  
when designing primers and probes for the mpcr / rlb assay it is strongly advised to use in silico methods to predict pcr products and probe hybridization to correlate with the in vitro results . ideally this is done using isolates for which the whole genome sequence is available .
software such as fastpcr ( available at http://primerdigital.com/fastpcr.html ) can be used for this purpose .
weak or absent probe signal can be predicted where in silico analysis indicates base pair mismatches resulting in low probe annealing temperatures .
dna extraction techniques will vary depending on the samples being tested , and pcr conditions will be dependent on primer design .
readers are referred to publications on individual assays for more information regarding dna extraction and pcr reagents and conditions .  
each assay is run with appropriate controls to provide at least one positive and one negative probe signal for each probe on the membrane , as well as a dna - free control .
furthermore it is beneficial to include a probe at the top of the membrane that is expected to be positive for all samples ( eg : a species or genus specific probe in the case of a micro - organism ) .
this serves as a positive control probe , but also permits easy orientation of the results .
prepare the following solutions : 100ml 0.5 m nahco3 ( ph 8.4)100 ml 0.5 m nahco320 ml 16% edac250 ml 0.1
clean the immunetics miniblotter with 70% ethanol . dilute the oligonucleotide probes in 0.5 m nahco3 to a final concentration of 2 pmol/l and a volume of 200 l .
seal membrane in plastic bag with 20 ml freshly made 16% edac solution and rock at room temperature for 10 minutes .
place membrane in miniblotter with channels running across the membrane ( parallel to pencil line ) . put support cushion in place and close blotter .
use 150 l of each probe solution to fill lanes 2 - 44 in sequence , being careful to avoid air bubbles .
if an air bubble does appear in the channel , keeping the pipette in place , rapidly aspirate the solution to allow the air bubble to float to the top of the pipette , then retry .
aspirate probe solutions , remove membrane and wash in 250 ml 0.1 m naoh at room temperature for 9 minutes .
wash membrane in 250 ml pre - warmed 2xsspe/0.1% sds in oven at 60c for 5 minutes .
if not being used for hybridization immediately , wash membrane in 240 ml 20 mm edta at room temperature for 20 minutes ( reserving remaining 10ml ) , then seal membrane in plastic bag with remaining 10 ml 20 mm edta and store in refrigerator at 4c .
prepare the following solutions : 250 ml 2xsspe/0.1% sds - store in water bath at 60c.500 ml 2xsspe/0.5% sds  store in water bath at 60c500 ml 2xsspe/0.5% sds  store in oven at 42c500 ml 2xsspe - keep at room temperature500 ml 1% sds  store in water bath at 60c initially ( for step 3)250 ml 20 mm edta 
sds  store in oven at 42c 500 ml 2xsspe - keep at room temperature 500 ml
1% sds  store in water bath at 60c initially ( for step 3 ) 250 ml 20 mm edta  store at room temperature ( for step 3 ) .
turn on one oven at 60c and one oven to 42c . bring water to boil in a large beaker on a hotplate .
add 20 l of each pcr product to 150 l 2xsspe/0.1% sds in numbered tubes . boil pcr products for 10 min at 100c using styrofoam holders .
add boiled pcr products to remaining channels ( 2 - 44 ) in sequence , being careful to avoid air bubbles .
place miniblotter flat in 60c oven for 1 hour to allow hybridization to take place .
moisten nylon separating mesh with 2xsspe/0.5% sds at 42c and use it to roll up membrane .
place rolled up membrane into roller tube , and unfurl to ensure membrane abuts the interior surface .
add 3 l streptavidin - peroxidase conjugate ( roche applied science ) to 15 ml 2xsspe/0.5% sds at 42c and add to roller tube .
turn oven up to 80c and place 500ml 1% sds in oven to pre - warm .
make up 15 ml amersham ecl detection solution ( 7.5 ml of solution a and 7.5 ml of solution b ) .
dog - ear lower right corner of film upon removal to allow correct orientation when viewing .
seal membrane in plastic bag with 10 ml 20 mm edta and refrigerate at 4c for future re - use .
the results are best viewed by placing the film over a printed grid , or else scanning the image and importing into software such as bionumerics ( applied maths , sint - martens - latem , belgium ) .
positive results are where the signal is as strong as or stronger than the positive control probe .
negative results are where the signal is absent or equal to the negative control ( in the case of background signal ) .
weak results are where the signal is fainter than the positive control probe , but stronger than the negative control . weak results may be the result of point mutations leading to weak probe binding , or due to non - specific signals from primer dimer formation .
using two probes per target of interest can improve the specificity , where a single weak result can safely be interpreted as non - specific signal .
if any doubt remains , a single - plex pcr reaction can be performed with gel - based detection and sequencing of any amplified product to determine if the result is truly positive .
streptavidin , labeled with peroxidase , is incubated with the membrane and binds to biotin .
peroxidase catalyses a reaction in the ecl detection reagents , producing light to which light sensitive film is exposed . the membrane is then washed for re - use .  
each target sequence ( a through u ) is detected with two different probes to maximize specificity .
probes a1 and a2 represent species - specific probes which are expected to be positive for each sample and assist with orienting the film .
samples 7 and 8 are negative controls . note that probes q1 , q2 , t1 and t2 have signal in the negative controls , indicating likely nonspecific binding of primers or primer dimer product . re - design of these probes or primers would be required .
probes c1 , d1 and f1 show streaking across the membrane likely due to some non - specific uptake of the streptavidin - peroxidase conjugate or chemiluminescent substrate , however this is easily distinguished from true positive probe signals .
probes d1 and d2 have relatively weak signal compared to other probes , this may be due to larger amplicons leading to less efficient amplification .
probe j2 is negative is several samples ( 1 , 3 , 4 , 6 , 39 ) where probe j1 is positive .
prepare the following solutions : 100ml 0.5 m nahco3 ( ph 8.4)100 ml 0.5 m nahco320 ml 16% edac250 ml 0.1 m naoh250 ml 2xsspe250 ml 2xsspe/0.1% sds  place in waterbath at 60c250 ml 20 mm edta .
100ml 0.5 m nahco3 ( ph 8.4 ) 250 ml 2xsspe/0.1% sds  place in waterbath at 60c pre - heat oven to 60c . clean the immunetics miniblotter with 70% ethanol . dilute the oligonucleotide probes in 0.5 m nahco3 to a final concentration of 2 pmol/l and a volume of 200 l . cut a biodynec nylon membrane to 15x15 cm .
using a pencil and ruler , rule off a 0.5 cm space across top of membrane and write details here .
seal membrane in plastic bag with 20 ml freshly made 16% edac solution and rock at room temperature for 10 minutes .
place membrane in miniblotter with channels running across the membrane ( parallel to pencil line ) . put support cushion in place and close blotter .
use 150 l of each probe solution to fill lanes 2 - 44 in sequence , being careful to avoid air bubbles . if an air bubble does appear in the channel , keeping the pipette in place , rapidly aspirate the solution to allow the air bubble to float to the top of the pipette , then retry .
aspirate probe solutions , remove membrane and wash in 250 ml 0.1 m naoh at room temperature for 9 minutes .
wash membrane in 250 ml pre - warmed 2xsspe/0.1% sds in oven at 60c for 5 minutes .
if not being used for hybridization immediately , wash membrane in 240 ml 20 mm edta at room temperature for 20 minutes ( reserving remaining 10ml ) , then seal membrane in plastic bag with remaining 10 ml 20 mm edta and store in refrigerator at 4c .
prepare the following solutions : 250 ml 2xsspe/0.1% sds - store in water bath at 60c.500 ml 2xsspe/0.5% sds  store in water bath at 60c500 ml 2xsspe/0.5% sds  store in oven at 42c500 ml 2xsspe - keep at room temperature500 ml 1% sds  store in water bath at 60c initially ( for step 3)250 ml 20 mm edta  store at room temperature ( for step 3 ) . 
 
sds  store in oven at 42c 500 ml 2xsspe - keep at room temperature 500 ml 1% sds  store in water bath at 60c initially ( for step 3 ) 250 ml 20 mm edta 
turn on one oven at 60c and one oven to 42c . bring water to boil in a large beaker on a hotplate .
add 20 l of each pcr product to 150 l 2xsspe/0.1% sds in numbered tubes .
add boiled pcr products to remaining channels ( 2 - 44 ) in sequence , being careful to avoid air bubbles .
place miniblotter flat in 60c oven for 1 hour to allow hybridization to take place .
moisten nylon separating mesh with 2xsspe/0.5% sds at 42c and use it to roll up membrane .
place rolled up membrane into roller tube , and unfurl to ensure membrane abuts the interior surface .
add 3 l streptavidin - peroxidase conjugate ( roche applied science ) to 15 ml 2xsspe/0.5% sds at 42c and add to roller tube .
turn oven up to 80c and place 500ml 1% sds in oven to pre - warm .
make up 15 ml amersham ecl detection solution ( 7.5 ml of solution a and 7.5 ml of solution b ) .
dog - ear lower right corner of film upon removal to allow correct orientation when viewing .
seal membrane in plastic bag with 10 ml 20 mm edta and refrigerate at 4c for future re - use .
the results are best viewed by placing the film over a printed grid , or else scanning the image and importing into software such as bionumerics ( applied maths , sint - martens - latem , belgium ) .
positive results are where the signal is as strong as or stronger than the positive control probe .
negative results are where the signal is absent or equal to the negative control ( in the case of background signal ) .
weak results are where the signal is fainter than the positive control probe , but stronger than the negative control .
weak results may be the result of point mutations leading to weak probe binding , or due to non - specific signals from primer dimer formation .
using two probes per target of interest can improve the specificity , where a single weak result can safely be interpreted as non - specific signal .
if any doubt remains , a single - plex pcr reaction can be performed with gel - based detection and sequencing of any amplified product to determine if the result is truly positive .
streptavidin , labeled with peroxidase , is incubated with the membrane and binds to biotin .
peroxidase catalyses a reaction in the ecl detection reagents , producing light to which light sensitive film is exposed . the membrane is then washed for re - use .  
samples 1 through 6 represent positive controls  between them there is at least one positive probe signal for each of the 43 probes .
each target sequence ( a through u ) is detected with two different probes to maximize specificity .
probes a1 and a2 represent species - specific probes which are expected to be positive for each sample and assist with orienting the film .
samples 7 and 8 are negative controls . note that probes q1 , q2 , t1 and t2 have signal in the negative controls , indicating likely nonspecific binding of primers or primer dimer product . re - design of these probes or primers would be required .
probes c1 , d1 and f1 show streaking across the membrane likely due to some non - specific uptake of the streptavidin - peroxidase conjugate or chemiluminescent substrate , however this is easily distinguished from true positive probe signals .
probes d1 and d2 have relatively weak signal compared to other probes , this may be due to larger amplicons leading to less efficient amplification .
probe j2 is negative is several samples ( 1 , 3 , 4 , 6 , 39 ) where probe j1 is positive .
the mpcr / rlb method permits simultaneous detection of a large number of pcr amplicons . because of the high sensitivity of chemiluminescent probe - based detection ,
a single multiplex pcr reaction with large numbers of primer pairs can be used to amplify the dna template . if no probe signals are obtained with one or more samples , performing gel electrophoresis with any remaining pcr product can help distinguish whether the problem is with pcr amplification or probe hybridization . where all probe signals on the membrane are weak or absent , but gel electrophoresis indicates successful pcr amplification , possibilities include problems with labeling of the membrane , incorrect regeneration of the membrane after previous use , incorrect temperatures during hybridization or streptavidin incubation , or defective streptavidin - peroxidase conjugate or detection reagent . if control samples indicate that individual probes may be producing false negative signals , single pcr with gel - based detection and subsequent sequencing can be performed to verify amplification of pcr product and to look for sequence variation at the probe binding site . weak or absent probe signal may be due to large amplicon size : if possible all amplicons should be of similar length and less than 300 bp .
secondary dna structures of amplicons may also produce poor probe binding , and may necessitate re - design of primers . individual primer or probe concentrations
false positive signals due to probe binding of nonamplified primers or primer - dimer product can be investigated by performing single pcr with gel - based detection and subsequent sequencing .
this characteristic may be exploited with careful primer and probe design to detect small sequence variations in target dna . in summary , while there are many methods of product detection following multiplex pcr reactions available , mpcr / rlb has the advantage of being high - throughput and inexpensive with low setup - costs .
the flexibility of the method permits its use in for a wide range of applications .
 multiplex pcr / reverse line blot hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex pcr reaction followed by probe hybridization on a nylon membrane , which is re - usable . 
 probes are 5 ' amine modified to allow fixation to the membrane . 
 primers are 5 ' biotin modified which allows detection of hybridized pcr products using streptavidin - peroxidase and a chemiluminescent substrate via photosensitive film . with low setup and consumable costs , this technique is inexpensive ( approximately us$2 per sample ) , high throughput ( multiple membranes can be processed simultaneously ) and has a short turnaround time ( approximately 10 hours).the technique can be utilized in a number of ways . 
 multiple probes can be designed to detect sequence variation within a single amplified product , or multiple products can be amplified simultaneously , with one ( or more ) probes used for subsequent detection . 
 a combination of both approaches can also be used within a single assay . 
 the ability to include multiple probes for a single target sequence makes the assay highly specific.published applications of mpcr / rlb include detection of antibiotic resistance genes1,2 , typing of methicillin - resistant staphylococcus aureus3 - 5 and salmonella sp6 , molecular serotyping of streptococcus pneumoniae7,8 , streptococcus agalactiae9 and enteroviruses10,11 , identification of mycobacterium sp12 , detection of genital13 - 15 and respiratory tract16 and other17 pathogens and detection and identification of mollicutes18 . 
 however , the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms.the five steps in mpcr / rlb are a ) primer and probe design , b ) dna extraction and pcr amplification c ) preparation of the membrane , d ) hybridization and detection , and e ) regeneration of the membrane . 
Protocol
1. Preparation of Membrane
2. Hybridization and Detection
3. Regeneration of Membrane
4. Representative Results:
Discussion
Disclosures