Contrast of Real-Time Fluorescent PCR Methods for Detection of Escherichia coli O157:H7 and of Introducing an Internal Amplification Control

Microorganisms
Xihong ZhaoYao Liu

Abstract

Various constituents in food specimens can inhibit the PCR assay and lead to false-negative results. An internal amplification control was employed to monitor the presence of false-negative results in PCR amplification. In this study, the objectives were to compare the real-time PCR-based method by introducing a competitive internal amplification control (IAC) for the detection of Escherichia O157:H7 with respect to the specificity of the primers and probes, analytical sensitivity, and detection limits of contamination-simulated drinking water. Additionally, we optimized the real-time fluorescent PCR detection system for E. coli O157:H7. The specificity of primers and probes designed for the rfbE gene was evaluated using four kinds of bacterial strains, including E. coli O157:H7, Staphylococcus aureus, Salmonella and Listeria monocytogenes strains. The real time PCR assay unambiguously distinguished the E. coli O157:H7 strains after 16 cycles. Simultaneously, the lowest detection limit for E. coli O157:H7 in water samples introducing the IAC was 104 CFU/mL. The analytical sensitivity in water samples had no influence on the detection limit compared with that of pure cultures. The inclusion of an internal amplification control i...Continue Reading

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Citations

Mar 21, 2020·Microorganisms·Xihong ZhaoTian Ding

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Methods Mentioned

BETA
PCR
electrophoresis
Fluorescence

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