Saito M, Matsumoto M, Funabashi M
Aichi Prefectural Institute of Public Health, Japan.
Int J Food Microbiol. 1992 Sep;17(1):47-55. doi: 10.1016/0168-1605(92)90018-x.
The polymerase chain reaction (PCR) procedure was evaluated to see if it is a simple and rapid method to detect Clostridium perfringens enterotoxin gene. The method, involving the use of two synthesised primers and gene amplification by the PCR procedure, detects a DNA fragment of 364 base pairs of C. perfringens enterotoxin gene by gel electrophoresis. The enterotoxin gene of strains was detected by use of purified chromosomal DNA. The supernatant of sporulating cultures in a sporulating medium was able to be used as template DNA. Template DNA can be obtained by merely culturing the strain in DS medium, a sporulating medium, for 48 h at 37 degrees C. All C. perfringens strains showing positive results in the PCR procedure were demonstrated to produce enterotoxin by a conventional method and all strains showing negative results were enterotoxin negative. To detect the enterotoxin gene in stool specimens by the PCR procedure, the specimen was heat-treated for 10 min at 90 degrees C and cultured in a sporulating medium, the supernatant of which was used as template DNA. From the stool specimens showing positive results in the PCR procedure by this method, enterotoxigenic C. perfringens was isolated from the heat-treated specimens. Thus, it is possible to detect enterotoxigenic C. perfringens in stools without isolation of the organism.
对聚合酶链反应（PCR）程序进行了评估，以确定它是否是检测产气荚膜梭菌肠毒素基因的一种简单快速的方法。该方法使用两条合成引物并通过PCR程序进行基因扩增，通过凝胶电泳检测产气荚膜梭菌肠毒素基因的一个364个碱基对的DNA片段。使用纯化的染色体DNA检测菌株的肠毒素基因。芽孢形成培养基中芽孢形成培养物的上清液可用作模板DNA。仅通过在芽孢形成培养基DS培养基中于37℃培养菌株48小时即可获得模板DNA。所有在PCR程序中显示阳性结果的产气荚膜梭菌菌株经传统方法证明可产生肠毒素，而所有显示阴性结果的菌株均为肠毒素阴性。为了通过PCR程序检测粪便标本中的肠毒素基因，将标本在90℃热处理10分钟并在芽孢形成培养基中培养，其上清液用作模板DNA。通过该方法在PCR程序中显示阳性结果的粪便标本中，从热处理后的标本中分离出产肠毒素产气荚膜梭菌。因此，无需分离该生物体即可检测粪便中产肠毒素产气荚膜梭菌。
