交叉引物扩增法检测食品中大肠杆菌不耐热肠毒素的研究

Detection of Heat-labile Enterotoxins of Escherichia coli in Food by Cross-primer Amplification

产肠毒素性大肠杆菌(enterotoxigenic Escherichia coli,ETEC)可引起严重食源性疾病,建立快速、准确的检测方法对保障食品安全和人类健康具有重要意义。对ETEC不耐热肠毒素(heat-labile enterotoxin,LT)基因保守序列设计交叉引物等温扩增(cross priming amplification,CPA)特异性引物,同时对反应温度、脱氧核糖核苷三磷酸(deoxyribonucleoside triphosphate,dNTPs)浓度、Bst-DNA聚合酶大片段浓度、Mg^(2+)浓度、甜菜碱浓度和反应时间进行优化;选取6株大肠杆菌和其他8株近源菌对CPA法进行特异性和敏感性分析;将纯培养后的产肠毒素大肠杆菌菌液稀释后随机污染46份不含LT肠毒素的生猪肉样品以评价该方法的应用效果。结果表明,CPA的最优反应温度为62℃、dNTPs浓度为0.3 mol/L、Bst聚合酶浓度为8 U/管、Mg^(2+)浓度为2.0 mmol/L、甜菜碱浓度为0.8 mol/L、反应时间为45 min;CPA方法检测产肠毒素大肠杆菌菌液的最低检出量为40 cfu/mL,为环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法的2倍,且特异性较高;46份人工污染生猪肉样品中共检测出阳性样品7份,CPA扩增结果与LAMP和荧光定量聚合酶链反应结果一致,检出率均为15.2%。

Enterotoxigenic Escherichia coli (ETEC) can cause serious foodborne diseases. It is important to establish a rapid and accurate detection method for food safety and human health. Cross-priming amplification (CPA) primers were designed based on the conservative sequence of ETEC heat-labile enterotoxin (LT) gene, and the reaction temperature, deoxy-ribonucleoside triphosphate (dNTPs) concentration, Bst-DNA polymerase-large fragment concentration, Mg^2+ concentration, betaine concentration and reaction time were optimized. Six strains of Escherichia coli and eight other near-source bacteria were selected to be specific for CPA method. In order to evaluate the application technology of CPA, 46 samples of raw pork without LT enterotoxin were randomly contaminated by diluted enterotoxin-producing Escherichia coli. The results showed that the optimum reaction temperature of CPA was 62 ℃, the concentration of dNTPs was 0.3 mol/L, the concentration of Bst-DNA polymerase-large fragment concentration was 8 U/tube, the concentration of Mg^2+ was 2.0 mmol/L, the concentration of betaine was 0.8 mol/L, and the reaction time was 45 mins. The minimum detection rate of enterotoxigenic Escherichia coli by CPA was 40 cfu/mL, which was two times higher than loop-mediated isothermal amplification (LAMP). The results of CPA assay in 7 positive samples (15.2 %) were consistent with those of LAMP and real-time fluorescence quantitative PCR.