LAMP实时浊度法快速检测食品中产志贺毒素大肠埃希氏菌

针对产志贺毒素大肠埃希氏菌(Shiga toxin-producing Escherichia coli,STEC)stx1和stx2基因的特异性序列分别设计4条特异性引物,采用环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术,利用实时浊度仪建立食品中STEC的LAMP实时浊度法快速检测方法。LAMP反应在实时浊度仪63℃恒温下1 h可完成。对方法的特异性、灵敏度、稳定性进行了评价,并在实际样品检测中进行应用。经优化该方法的检测灵敏度可达150拷贝/反应,4株STEC菌株LAMP扩增结果与其基因型一致,其他21株非STEC菌株均未出现非特异性扩增。395份食品样品检出STEC阳性68份(阳性率为17.2%),所检食品类别中畜产品阳性率最高(达31.3%),禽产品、水产品和可生食蔬菜均有少量阳性样品检出,阳性样品中以stx2基因阳性型为主。结果表明,LAMP实时浊度法具有快速、灵敏、特异性强、操作简便的优势,适用于食品中STEC的快速筛查。

Rapid Detection of Shiga Toxin-Producing Escherichia coli by LAMP Real-Time Turbidity Method

Four specific primers were designed for each of the specific sequences of stx1 and stx2 gene of shiga toxin-producing Escherichia coli(STEC). Real-time monitoring of the loop-mediated isothermal amplification(LAMP)reaction was realized with the turbidimeter. Detection could be completed at 63℃ on the real-time turbidimeter in 1 h. The specificity,sensitivity,stability of the method was evaluated,and the method was applied in food detection. The detection limit of the method was 150copies per reaction mixturein optimized condition,and the LAMP amplification results of 4 STEC strains were compliant with their own genetypes separately and 21 non-STEC strains showed no unspecific amplification in the meantime. 68 STEC positive samples was detected in 395 food samples with the positive ratio of 17.2%,the positive ratio oflivestock products was 31.3% which was the highest in all food types,and lower positive ratio were also detected in poultry products,aquatic products andedible raw vegetables. Samples carried stx2 genesignificantly more in positive samples.It suggested that the LAMP real-time turbidity method was rapid,sensitive,high specificity and easy to operate,and could be used as a fast screening method for STEC in food.