动物性食品中大肠埃希氏菌O157∶H7特异性二重PCR检测方法的建立

根据大肠埃希氏菌O157∶H7wzx和fliC基因的保守序列设计了2对引物,建立并优化了特异性检测大肠埃希氏菌O157∶H7的二重PCR体系,扩增产物为395bp(wzx)和1057bp(fliC)。人工培养基中大肠埃希氏菌O157∶H7的检测下限为2.3×102CFU/mL,人工污染的牛乳、猪肉及牛肉中大肠埃希氏菌O157∶H7的检测下限分别为5.8×102CFU/mL、4.3×102CFU/g和3.1×103CFU/g。样品经3h预增菌后检测下限可达3.1×100~5.8×100CFU/mL(或CFU/g)。试验结果提示,针对wzx及fliC基因的二重PCR方法可以快速特异地检测出牛乳、猪肉及牛肉中大肠埃希氏菌O157∶H7的污染。

Development of duplex PCR for detecting Escherichia coli O157∶H7 in foods of animal origin

A duplex PCR procedure was developed for specific detection of Escherichia coli O157H7 based on its specific wzx and fliC genes. Fragments of 1057bp(fliC) and 395bp(wzx) could be amplified only from E. coli O157H7, but not from other bacterial species by the PCR. The detection limit was (2.310~(2))CFU/mL in artificial broth medium, or 5.810~(2)CFU/mL, 4.310~(2)CFU/g and 3.110~(3)CFU/g in artificially-contaminated milk, pork meat and beef respectively. The detection limit could be improved to 3.110~0~5.810~(0)CFU/mL(or CFU/g) in such contaminated samples if combined with further incubation at 37 for 3h. This PCR procedure is rapid, specific and sensitive for detection of E.coli (O157H7) in foods of animal origin such as pork, beef and milk.