环介导等温核酸扩增技术快速检测食物中的大肠杆菌O157

Development and Application of a Loop-Mediated Isothermal Amplification Method on Rapid Detection Escherichia coli O157 Strains from Food Samples

建立了环介导等温核酸扩增技术(LAMP)检测食源大肠杆菌O157,并对该方法的灵敏度和特异性进行了评价。分别针对大肠杆菌O157三个特异基因rfbE,stx1和stx2的8个独立靶区域设计了外引物、内引物和环引物进行LAMP扩增检测,同时将检测结果与PCR方法做比较。研究结果表明,rfbE,stx1和stx2基因的LAMP方法检测限分别为100,100和10 fg DNA/管,灵敏度是PCR方法的10倍以上;将建立的环介导等温扩增法用于417株食物分离的大肠杆菌的检测,发现LAMP检测rfbE,stx1和stx2基因的灵敏度分别为100%,95.3%和96.3%,对3个靶基因的阴性预测率分别为100%,96.7%和97.1%,特异性和阳性预测率均为100%。结果表明,该方法用于大肠杆菌O157的检测具有特异性强、灵敏度高、操作简便的优越性,在食品安全检测方面具有良好的实际应用前景。

The specificity and sensitivity of a loop-mediated isothermal amplification （LAMP） method was developed and evaluated for rapid detection of the food-borne Escherichia coli O157 strains. Six primers, including outer primers, inner primers and loop primers, were specially designed for recognizing eight distinct sequences on three targets, which were rfbE, stxl and stx2. The detection limits were found to be 100, 100 and 10 fg DNA/tube for rfbE, stxl and stx2, respectively. Application of LAMP assays were performed on 417 food-borne E. coli strains, the sensitivity of LAMP assays for the rfbE, stxl and stx2 was 100% , 95.3% and 96.3% , and the negative predictive value （NPV） was 100%, 96.7% and 97.1% respectively; with a 100% specificity and positive predictive value （PPV） for all three targets. The results showed that the LAMP assay has proven high specificity, sensitivity and easy operation features in the detection of Escherichia coli 0157. It also implied that the assay owned good application prospect in food safety detection.