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

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

根据大肠埃希氏菌O157∶H7 wzx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.

A duplex PCR procedure was developed for specific detection of Escherichia coli O157 ： H7 based on its specific wzx and fliC genes. Fragments of 1 057 bp（fliC） and 395 bp（wzx） could be amplified only from E. coli O157 ： H7, but not from other bacterial species by the PCR. The detection limit was 2.3X102 CFU/mL in artificial broth medium, or 5. 8 × 10^2 CFU/mL, 4. 3 × 10^2 CFU/g and 3. 1 × 10^3 CFU/g in artificially-contaminated milk, pork meat and beef respectively. The detection limit could be improved to 3.1 × 10^0～5.8 × 10^0 CFU/mL（or CFU/g） in such contaminated samples if combined with further incubation at 37℃ for 3 h. This PCR procedure is rapid, specific and sensitive for detection of E. coli O157 ： H7 in foods of animal origin such as pork, beef and milk.