Construction and primary application of a TAQMAN PCR detection method for detection of Escherichia coli O₁₅₇H_7

Objective To establish a rapid sensitive and specific detection method of Escherichia coli O₁₅₇:H₇ with TaqMan PCR.Methods To design a pair of primers and probe depending on rfbE gene by way of target sequence of Escherichia coli O₁₅₇:H₇, and apply Escherichia coli O₁₅₇:H₇ of standard bacterium strain for template to appraise Escherichia coli O₁₅₇:H₇.The best Mg²⁺ concentration.primer and probe ratio were optimized.Specificity, sensitivity and stability analysis test were performed by Escherichia coli O₁₅₇:H₇ and 10 other associated bacteria strains.Results The best Mg²⁺ concentration was 4mmoL/L.Concentretion of primers and probe was 0.6μmoL/L and 0.8μmoL/L.Tests showed that the probe were highly conservative and specific.The results of all 10 bacterial strains were negative except of strain of Escherichia coli O₁₅₇:H₇.The quantitative detection limit of the method was 17cfu/mL in pure broth culture.Stability test showed that coefficient variables were all less than 5% in 4 different concentrations.The results showed that real-time PCR method was more sensitive, easier and faster than conventional culture method for detection of Escherichia coli O₁₅₇:H₇.Conclusion Real-time PCR method has high sensitivity and specificilty.The real-time PCR is a handy and rapid method.It can be used in examination of microorganisms in food and has great value in practical work.