A Method for the Detection of Escherichia coli O157 Based on Real-time Fluorescence Single Primer Isothermal Amplification (SPIA)
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Abstract:
The aim of this study was to establish a rapid, sensitive method for detecting enterohemorrhagic Escherichia coli O157 in foods. To establish a real-time fluorescence single primer isothermal amplification (real-time fluorescence SPIA) method for the detection of Escherichia coli O157, an RNA/DNA primer and chain terminator were designed based on the sequence of the rfbE gene of Escherichia coli O157, and the reaction conditions, such as primer concentration, Mg2+ concentration, and temperature, were optimized. The specificity, sensitivity, and detection limit for artificial contamination were determined. Under optimal conditions, a sample measurement could be completed within 55 min. No other bacteria besides E. coli O157 showed a specific fluorescent amplification curve. The sensitivity of the real-time fluorescence SPIA method for the detection of E. coli O157 was 2.0 CFU/mL, and the detection limit for an artificially contaminated pork sample was 4.0 CFU/g. The advantages of this real-time fluorescence SPIA assay include short detection time, high sensitivity, high specificity, and convenience. This study establishes a technology platform for the detection of food-borne pathogens.
