Meer R R, Songer J G
Department of Veterinary Science, University of Arizona, Tucson 85721, USA.
Am J Vet Res. 1997 Jul;58(7):702-5.
To develop a multiplex polymerase chain reaction (PCR) assay to detect the genes for the major toxins of Clostridium perfringens (cpa [alpha toxin], cpb [beta toxin], etx [epsilon toxin], iA [iota toxin], and cpe [enterotoxin]).
Cultures of C perfringens obtained from collections and diagnosticians throughout North America.
PCR primers were derived from published sequences of the genes for the major toxins (the "typing" toxins and enterotoxin). The concentration of each primer was titrated in a PCR assay to allow concurrent amplification of multiple target sequences, and other parameters of the assay were optimized (including concentrations of other reagents and times and temperatures for denaturation of template, annealing of primers, and primer extension). Specificity of the assay was measured by comparing genotype with phenotype (where it was known).
The genotype, determined by multiplex PCR assay, agreed with phenotype in 99% (86/87) of strains where phenotype had been determined. Applied to 361 isolates from domestic animals and human beings, 95% (n = 344) were type A, and 12.8% (n = 44) of these contained cpe. The remaining 5% (n = 17) of the isolates were type B (n = 1), type C (n = 11), type D (n = 2), or type E (n = 4).
Previous studies have documented usefulness of PCR in genotyping C perfringens. The multiplex assay is as effective, but simpler, and may be a useful alternative to standard in vivo typing methods. Results of genotyping of field isolates suggested the need for further epidemiologic study of clostridial enteritis, particularly as this pertains to predominant etiologic toxin types, and documented the presence of the reportedly rare genotypes B and E.
开发一种多重聚合酶链反应（PCR）检测方法，以检测产气荚膜梭菌主要毒素的基因（cpa[α毒素]、cpb[β毒素]、etx[ε毒素]、iA[ι毒素]和cpe[肠毒素]）。
从北美各地的收藏机构和诊断人员处获得的产气荚膜梭菌培养物。
PCR引物源自主要毒素（“分型”毒素和肠毒素）基因的已发表序列。在PCR检测中对每种引物的浓度进行滴定，以实现多个靶序列的同时扩增，并对检测的其他参数进行优化（包括其他试剂的浓度以及模板变性、引物退火和引物延伸的时间和温度）。通过比较基因型与表型（已知时）来测定检测的特异性。
在已确定表型的菌株中，多重PCR检测确定的基因型与表型在99%（86/87）的菌株中一致。应用于361株来自家畜和人类的分离株，95%（n = 344）为A型，其中12.8%（n = 44）含有cpe。其余5%（n = 17）的分离株为B型（n = 1）、C型（n = 11）、D型（n = 2）或E型（n = 4）。
先前的研究已证明PCR在产气荚膜梭菌基因分型中的有用性。多重检测同样有效，但更简单，可能是标准体内分型方法的一种有用替代方法。现场分离株的基因分型结果表明需要对梭菌性肠炎进行进一步的流行病学研究，特别是关于主要病因毒素类型方面，并证明了据报道罕见的B型和E型基因型的存在。
