MULTIPLEX POLYMERASE CHAIN-REACTION ASSAY FOR GENOTYPING CLOSTRIDIUM-PERFRINGENS

Objective-To develop a multiplex polymerase chain reaction (PCR) assay to detect the genes for the major toxins of Clostridium perfringens ( cpa [ct toxin], cpb [beta toxin], elx [epsilon toxin], iA [iota toxin] , and cpe [enterotoxin]). Sample Population-Cultures of C perfringens obtained from collections and diagnosticians throughout North America. Procedure-PCR primers were derived from published sequences of the ge nes for the major toxins (the ''typing'' toxins and enterotoxin). The concentration of each primer was titrated in a PCR assay to allow conc urrent amplification of multiple target sequences, and other parameter s of the assay were optimized (including concentrations of other reage nts and times and temperatures for denaturation of template, annealing of primers, and primer extension). Specificity of the assay was measu red by comparing genotype with phenotype (where it was known). Results -The genotype, determined by multiplex PCR assay, agreed with phenotyp e in 99% (86/87) of strains where phenotype had been determined. Appli ed to 361 isolates from domestic animals and human beings, 95% (n = 34 4) were type A, and 12.8% (n = 44) of these contained cpe. The remaini ng 5% (n = 17) of the isolates were type B (n = 1), type C (n = 11), t ype D (n = 2), or type E (n = 4).Conclusion and Clinical Relevance-Pre vious studies have documented usefulness oi PCR in genotyping C perfri ngens. The multiplex assay is as effective, but simpler, and may be a useful alternative to standard in vivo typing methods. Results of geno typing of field isolates suggested the need for further epidemiologic study of clostridial enteritis, particularly as this pertains to predo minant etiologic toxin types, and documented the presence of the repor tedly rare genotypes B and E.