Miwa N, Nishina T, Kubo S, Fujikura K
Shizuoka Prefectural Western Meat Inspection Center, Japan.
J Vet Med Sci. 1996 Mar;58(3):197-203. doi: 10.1292/jvms.58.197.
A rapid and sensitive method for detecting enterotoxigenic Clostridium perfringens in animal feces and meat is described. The method consists of a combination of nested polymerase chain reaction (PCR) with enrichment culture of the sample. In the PCR, two pairs of oligonucleotide primers homologous to the C. perfringens enterotoxin (CPE) gene were used: the first primer pair amplified a 425-bp fragment and the second one amplified a 199-bp fragment within the fragment amplified by the first PCR. When the specificity and sensitivity of nested PCR in the detection of the CPE gene of isolated C. perfringens were compared with those of the single PCR, the former amplified specifically the 199-bp fragment and the sensitivity was about 10(3)-fold higher that that of the latter. The nested PCR combined with enrichment culture was applied to detecting the CPE gene in samples inoculated artificially with enterotoxigenic C. perfringens. This method detected within 22-26 hr the CPE gene in samples of animal feces and meat inoculated with fewer than 10(1) CFU/g of enterotoxigenic C. perfringens. When the method was applied to detection of indigenous enterotoxigenic C. perfringens in cattle feces, pig feces, beef and pork, C. perfringens was found in one case (10%) of 10 cattle fecal samples, and the PCR-amplified product corresponded to the fragment of the CPE gene in restriction endonucleases digestion pattern.
本文描述了一种快速、灵敏的检测动物粪便和肉类中产肠毒素性产气荚膜梭菌的方法。该方法将巢式聚合酶链反应（PCR）与样品的富集培养相结合。在PCR中，使用了两对与产气荚膜梭菌肠毒素（CPE）基因同源的寡核苷酸引物：第一对引物扩增出一个425 bp的片段，第二对引物在第一次PCR扩增的片段内扩增出一个199 bp的片段。将巢式PCR检测产气荚膜梭菌分离株CPE基因的特异性和敏感性与单重PCR进行比较时，前者特异性扩增出199 bp的片段，敏感性比后者高约10³倍。将巢式PCR与富集培养相结合，用于检测人工接种产肠毒素性产气荚膜梭菌的样品中的CPE基因。该方法在22 - 26小时内检测到接种量低于10¹ CFU/g产肠毒素性产气荚膜梭菌的动物粪便和肉类样品中的CPE基因。当该方法应用于检测牛粪便、猪粪便、牛肉和猪肉中的天然产肠毒素性产气荚膜梭菌时，在10份牛粪便样品中有1份（10%）检测到产气荚膜梭菌，PCR扩增产物在限制性内切酶消化图谱上与CPE基因片段相对应。
