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