Assembly of A Fluorescence-based Quantitative Real-time PCR Rapid Detection Kit for Viable Escherichia coli O157:H7
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Abstract:
A fluorescence-based quantitative real-time polymerase chain reaction (PCR) rapid detection kit for Escherichia coli O157: H7, developed based on high-resolution melt curve analysis, was assembled and evaluated for sensitivity, specificity, anti-interference ability, and detection of artificially contaminated samples. On the basis of kit assembly, we developed a quantitative detection technique for viable E. coli O157:H7 by utilizing the selectivity of propidium monoazide (PMAxx) for viable/dead cells. Our results indicated that the kit was highly specific, with positive amplification occurring only for the E. coli O157:H7 serotype, with a minimum detection limit of 84 CFU/mL. E. coli O157:H7 could be accurately detected under the interference of four common foodborne pathogens. While testing artificially contaminated food, samples with an initial contamination level of 7.97×100 CFU/mL could be detected. Subsequently, various parameters of the PMAxx treatment system were optimized by single-factor experiments and the following optimum reaction conditions were determined: exposure time: 11 min, dark incubation time: 20 min, PMAxx concentration: 30 μmol/L. By combining the optimized parameters with our assembled kit, we established a quantitative method for the detection of E. coli O157:H7 with a good linear relationship between Ct value and bacterial solution concentration within the concentration range of 3.4×107~3.4×102 CFU/mL. The results of this study provide a scientific basis for the rapid quantitative detection of foodborne pathogens.
