Taqman探针荧光PCR法检测食品中的大肠埃希氏菌O145

Detection of Escherichia coli O145 in Food by TaqMan probe Real-time PCR

摘要:

针对大肠埃希氏菌O145的O抗原基因簇的wckD基因的特异性序列设计引物和Taqman探针，建立检测大肠埃希氏菌O145的荧光PCR方法，对其灵敏度、特异性进行验证，并将其用于食品样品的检测。结果表明，本研究中的方法可实现对大肠埃希氏菌O145的特异性扩增，其它27株非O145大肠埃希氏菌和20株非大肠埃希氏菌细菌的菌株均无扩增；检测的灵敏度可达165拷贝/反应；339份食品样品EC肉汤增菌后用本荧光PCR法进行检测，检出大肠埃希氏菌O145阳性31份，阳性率为9.1%。实验结果表明，本研究成功建立了可用于食品中大肠埃希氏菌O145的Taqman探针荧光PCR方法，食品样品采用EC肉汤增菌24 h、热裂解法提取核酸，增菌后检测所需时间由至少3 d~7 d缩短为仅需2 h~3 h，食品检测全过程仅需约28 h，经证实本方法特异性强、操作简便，为食品中大肠埃希氏菌O145提供了一种的快速检测手段。

Abstract:

The primes and a TaqMan probe were designed to amplify the specific sequences of wckD gene in O-antigen gene group of Escherichia coli O145, and real-time PCR method was developed to detect Escherichia coli O145. The sensitivity and the specification of the method were verified, and the method was applied to the food detection. The results showed that E. coli O145 could be amplified specifically by the method, while the other 27 different strains of non-O145 E. coli and 20 different strains of non-Escherichia coli had not shown any amplification curve, and the test sensitivity could reach 165 copies for each reaction mixture. A total of 339 samples had been detected by the real-time PCR method after enrichment in EC broth, and 31 samples showed positive results with the positive ratio of 9.1%.Therefore, the TaqMan probe real-time PCR method for detection of E. coli O145 in food was successfully established,. Food samples were enriched with EC broth for 24 h and heat-lyse treatment was selected for the nucleic acid extraction, and the detection time was reduced from 3 d ~ 7 d to only 2 h ~ 3 h for post-enrichment samples, and the whole time for food detection only needed about 28h,.The method provides a rapid detection of E. coli O145 in food for its specificity and easy operation verified in this study.