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

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 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 O157:H7. The specificity of primers and probes designed for the gene was evaluated using four kinds of bacterial strains, including O157:H7, , and strains. The real time PCR assay unambiguously distinguished the O157:H7 strains after 16 cycles. Simultaneously, the lowest detection limit for O157:H7 in water samples introducing the IAC was 10 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 in the real-time PCR assay presented a positive IAC amplification signal in artificially simulated water samples. These results indicated that real-time fluorescent PCR combined with the IAC possessed good characteristics of stability, sensitivity, and specificity. Consequently, the adjusted methods have the potential to support the fast and sensitive detection of O157:H7, enabling accurate quantification and preventing false negative results in O157:H7 contaminated samples.