the disease necrotic enteritis ( ne ) is a major issue affecting the poultry industry , causing extensive economic loss through mortality , reduced bird performance , and carcass condemnation at slaughter [ 1 , 2 ] . caused by the bacterium clostridium perfringens ( cp ) , ne is characterized by necrotic lesions , primarily in the jejunum and ileum , which can vary in severity from thickened mucosa and multifocal ulceration in less severe cases to the formation of a greenish or yellowish pseudomembrane in the case of extensive mucosa inflammation and necrosis . in the past , alpha - toxin produced by cp type a has been implicated as the primary virulence factor in ne pathogenesis [ 2 , 4 , 5 ] ; however , the netb toxin , encoded by the netb gene , was shown to be important for the virulence of certain strains [ 6 , 7 ] .
another toxin , tpel ( encoded by the tpel gene ) , is a potential virulence factor as well . in a recent study , inoculation of broilers with strains positive for both tpel and netb
existing studies ascertaining the prevalence of netb and tpel have been limited to certain geographic populations of cp , mainly in australia , belgium , denmark , sweden , and canada [ 1 , 7 ] . in the united states , studies on the prevalence of these genes
the relatively small number of sampled cp populations underlines a need for more analysis to determine the importance of these genes on a worldwide scale .
additionally , detection of these genes has been performed relatively inefficiently using two single locus pcrs .
this report describes a multiplex pcr developed to detect netb and tpel simultaneously , providing a more efficient tool in screening cp strains for these genes .
songer of iowa state university ( jgs-1870 and jgs-4140 ) and the mitchem - sparks regional diagnostic laboratory located in boaz , alabama ( c103 - 99 ) .
all strains ( table 1 ) were prepared for dna extraction by enriching in cooked meat medium ( himedia laboratories pvt . ltd . , mumbai , india ) for 24  hr and then streaking the enriched samples for isolation onto trypticase soy agar with 5% sheep 's blood ( bd diagnostics , franklin lakes , nj ) .
after 2448  hr , a single colony exhibiting typical hemolysis was placed into brain - heart infusion ( bhi ) broth ( bd diagnostics ) and incubated for 24  hr . all incubation was carried out at 37c within a bactron iv anaerobic environmental chamber ( shel lab , cornelius , or ) with the following atmospheric conditions : 90% n2 , 5% co2 , and 5% h2 .
after incubation , bhi broth cultures were centrifuged at 16,000  g for 5  min and the broth supernatant was aspirated and discarded . utilizing a wizard genomic dna purification kit ( promega corp . ,
extracted dna was analyzed for quantity and purity with a nanodrop 1000 spectrophotometer ( thermo scientific inc .
a previously published primer set   was used for the detection of tpel and a primer set detecting netb was designed using the primer basic local alignment search tool ( blast ) function on the national center for biotechnology website .
the netb primers were confirmed to target mostly conserved sequences by performing a sequence alignment with 47 netb sequences in genbank using genetic analysis software .
econotaq plus green master mix ( lucigen corp . , middleton , wi ) was used at 1x concentration providing taq polymerase , mgcl2 , dntps , and reaction buffer ( ph 9.0 ) .
reagent concentrations of the optimized multiplex were as follows : 1.2  units/25  ul of taq dna polymerase ; 1.44  mm of mgcl2 ; 192  um of each dntp ; 0.2  um of each primer ; and 100  ng/25  ul of template dna .
the following cycling conditions were used : an initial denaturing step at 95c for 5  min ; 40 cycles of denaturing at 95c for 30  sec ; annealing at 55c for 30  sec , and extension at 72c for 30  sec , with a final extension step at 72c for 7  min .
gels and running buffer were made using 1x concentrations of accugene tbe buffer ( lonza group , basel , switzerland ) . for visualization of bands , 1  ul of an ethidium bromide solution ( 10  mg / ml ) was added to the molten gel before solidifying .
electrophoresis took place at 100  v for about one hr or until sufficient separation between products occurred .
a 100  bp dna ladder ( promega ) was utilized as a dna size standard .
bands corresponding to the expected amplicons were excised and dna was purified using a gfx pcr dna and gel band purification kit ( ge healthcare , buckinghamshire , uk ) . forward and
reverse strands of the purified dna were sequenced by lucigen and a consensus sequence was determined for each amplicon .
these consensus sequences were then subjected to a nucleotide blast search using the ncbi database nucleotide collection ( nr / nt ) to calculate the likelihood that each pcr product was produced from the target gene .
after the analysis of 47 netb gene sequences published in genbank , the target sequences for both forward ( netb5f ) and reverse ( netb5r ) primers proved to be mostly conserved .
a single polymorphism was present at site number 756 of the alignment , with 21 ( 44.7% ) of the sequences having g at this site and 26 ( 55.3% ) having a. this site was complementary to the sixth base from the 5 end of primer netb5r and resulted in a single a : c mismatch between the primer and 55.3% of the gene sequences .
this same mutation affects primers previously described by keyburn et al . , resulting in a g : t mismatch between the seventh base from the 5 end of akp78 ( the forward primer ) and 24 ( 51.1% ) of the analyzed gene sequences .
gel electrophoresis of pcr products confirmed the amplification of the target sequences for all three positive controls .
bands of the expected size corresponding to the netb ( 316  bp ) and tpel ( 466  bp ) fragments were observed for the positive controls , and no bands were observed for the negative controls .
sequences obtained from pcr products matched the target sequence ( at least 99% max identity ) with significantly low e - values after performing a blast search ( table 3 ) .
the multiplex pcr described in this report successfully detected the netb and tpel genes in three control organisms and did not produce false positives in the negative controls .
the chance of a false negative was minimized by choosing primers that targeted mostly conserved sites within the c. perfringens genome .
after analysis of 47 sequences , one polymorphism was observed in the target sequence indicating an internal a : c mismatch between the netb5r primer and 55.3% of the 47 aligned sequences .
although mismatches between a template and the 3 end of a primer are known to interfere with the active site of dna polymerase and can result in reduced reaction efficiency [ 11 , 12 ] , internal mismatches , or those closer to the 5 end , have a much smaller impact .
in addition , out of the possible mismatches , a : c ( purine - pyrimidine ) mismatches generally produce only minor interferences . previously published primer akp78 contained a g : t mismatch between 51.1% of the sequences .
as g : t is another purine - pyrimidine mismatch and the mismatch also occurred near the 5 end of the primer , it is comparable to netb5r . in consequence , it is unlikely that this mismatch between the template and netb5r would have a significant impact on product formation .
although evidence suggests the netb and tpel toxin genes may be important virulence factors for certain strains of c. perfringens , relatively few bacterial populations have been screened for these two genes .
more populations must be screened to determine the overall impact of these genes in ne . by pairing the detection of both netb and tpel into one pcr reaction
, this multiplex pcr has the potential to increase the efficiency with which strains are screened for these two genes of interest .
the effectiveness of this assay has been shown in three control organisms and must now be confirmed using a larger number of strains .
 some studies have shown that the netb toxin may be an important virulence factor of clostridium perfringens associated necrotic enteritis in poultry . additionally , research has shown that strains of c. perfringens positive for both the netb gene and a second toxin - encoding gene , tpel , appear to be more virulent than strains with only netb . in the past 
 , detection of these genes has been performed relatively inefficiently using two single locus pcrs . 
 this report describes a novel multiplex pcr developed to detect netb and tpel simultaneously in c. perfringens strains isolated from cases of necrotic enteritis in broilers , providing a more efficient diagnostic tool in the screening of strains for these genes . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion