实时荧光单引物等温扩增(SPIA)技术检测大肠杆菌O157的方法研究

建立一种快速、灵敏的食品中肠出血性大肠杆菌O157的检测方法。以大肠杆菌O157的特异性基因(rfbE)为靶序列,设计相应的RNA/DNA组合引物和链终止序列,优化引物浓度、Mg2+浓度、温度等反应条件,建立实时荧光单引物等温扩增(Real Time Fluorescence Single Primer Isothermal Amplification,实时荧光SPIA)检测大肠杆菌O157的方法,并对该方法的特异性、灵敏度以及人工污染检出限进行检测。确定了实时荧光SPIA法的最适反应条件,该方法可以在55 min内完成检测工作,除大肠杆菌O157外,其他细菌均未产生特异性荧光扩增曲线,而且实时荧光SPIA检测大肠杆菌O157的灵敏度为2.0 CFU/m L,对猪肉人工污染样品中大肠杆菌O157的检出限是4.0 CFU/g。实时荧光SPIA方法检测大肠杆菌O157具有耗时短,灵敏度高,特异性强,方法简便的优越性,为检测食源性致病菌构建了一个技术平台。

A Method for the Detection of Escherichia coli O157 Based on Real-time Fluorescence Single Primer Isothermal Amplification (SPIA)

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.