Study on Real-time PCR Detection Methods for Escherichia coli O157:H7

Objective To establish a sensitive,specific,and rapid method for detection of Escherichia coli O157: H7,and to apply it in the emergent public health events and epidemiological investigation of foodborne pathogenic bacteria. Methods According to the GenBank Escherichia coli O157: H7rfbE gene sequence, the primers and TaqMan probe were designed.The real-time fluorescent PCR reaction conditions were optimized.The real-time fluorescent PCR detection system for Escherichia coli O157: H7 was established.And the specificity,sensitivity and reproducibility of the method were evaluated. Results The test results of Escherichia coli O157: H7 strains were positive,whereas the test results of all other strains were negative.The sensitivity of the detection method was up to 1×102 cfu/ml.The simulated samples of pork,mutton,chicken,fresh vegetables were found to be contaminated with 1×104cfu/mL bacteria.The duration between nucleic acid extraction to bacteria detection was approximately 3 hours. Conclusions The established real-time fluorescence PCR detection method has the advantages of high sensitivity,strong specificity and rapidness,which can be used in Escherichia coli O157: H7 food poisoning diagnosis and a rapid microbiological testing of food.It provides a new detection way for the molecular epidemiological survey of foodborne diseases.