关键词: 大肠埃希氏菌O157∶H7, rfbE, Flic, 实时荧光定量PCR, 内参

Abstract:

Based on the rfbE and Flic genes of Escherichia coli O157:H7, the specific primers and probes were designed,
and a real-time fluorescence quantitative PCR (RT-qPCR) was developed. An internal amplification control (IAC) was
added to the reaction system to monitor the performance of reaction system. The assay could be used reliably to detect
E. coli O157:H7 genomic DNA with a sensitivity of 1 pg/μL. For the plasmid with rfbE and Flic, the limit of detection (LOD)
reached 103 copies/μL. The LOD for E. coli O157:H7 was 5 × 103 CFM/mL using the DNA extracted by water boiling as the
template. Through the standard curves of rfbE and Flic, the quantification was linear between Ct values and the copy number
of template (R2 = 0.999). For artificially contaminated meat samples with an initial bacterial concentration of 7 CFΜ/25 g,
the E. coli O157:H7 could be detected after 6 hours of culture using the DNA extracted by a commercial kit. Using the
DNA extracted through water boiling, the E. coli O157:H7 could be detected after 10 hours of culture. The fluorescence
quantitative PCR assay could be applied to detect E. coli O157:H7 in food samples and monitor the PCR reaction process
without false negative results. Furthermore, the comparison results of two different DNA extraction methods were helpful to
standardize the RT-qPCR method for E. coli O157:H7.

Key words: E.coli O157:H7, rfbE, Flic, real-time quantitative PCR, internal amplification control (IAC)