复合聚合酶链反应同时检测二个腹泻毒力因子

Detection of the thermostable direct hemolysin gene of vibrio parahaemolyticus and thermostable enterotoxin gene of enterotoxigenic escherichia coil by multi polymerase chain reaction

为检测腹泻病原菌产毒性大肠杆菌(ETEC)和副溶血弧菌的两个毒力因子基因热稳肠毒素和热稳直接溶血素,用标准模板建立复合聚合酶链反应(PCR),并检测了模拟标本和88份临床腹泻大便标本。结果热稳肠毒素和热稳直接溶血素基因引物扩增各自的标准模板分别产生186bp 和425bp 的片段,一元和复合 PCR 都只扩增热稳肠毒素和热稳直接溶血素基因 DNA 并产生特异片段,对其它细菌无扩增。热稳肠毒素和热稳直接溶血素的一元 PCR 扩增下限分别是80cfu 和40cfu,复合 PCR 分别是320cfu 和160cfu。88份腹泻标本,一元 PCR 检出副溶血弧菌的热稳直接溶血素和产毒性大肠杆菌的热稳肠毒素分别为1例和7例。而三份模拟标本则均被一元PCR 和复合 PCR 检出。结果提示复合 PCR 是快速、灵敏、特异和简便的检测 VP 和 ETEC 的方法。

Direct PCR and multi PCR protocols were established for specific detection of the ther- mostable direct hemolysin gene (tdh) and thermostable enterotoxin (st) genes as well as the respective virulense marker genes of vibrio parahaemolyticus (vp) and enterotoxigenic escherichia coil (ETEC). Their specificity was assessed with other diarrhea pathogens.Results showed that they were very specif- ic.The sensitivities of tdh and st direct PCR were 40cfu and 80cfu respectively,and multi PCR was less sensitive than direct PCR,which were 160cfu and 320cfu.The detection of three mimic samples con- taining different amount of vp(tdh+)and ETEC(st+) by multi PCR showed this method was reliable. In 88 clinical diarrhea samples detected by both direct PCR and multi PCR,7 were ETEC (st+) posi- tive and 1 was vp(tdh+) positive.The results of multi PCR were the same with direct PCR.Multi PCR was more convenient thin direct PCR,and was a rapid,simple,sensitive and specific method in the detection of vp (tdh+) and ETEC(st+).