食品中大肠埃希氏菌O121荧光PCR检测方法的研究

Research on a Real-time PCR Method for Detection of Escherichia coli O121 in Food

针对大肠埃希氏菌(Escherichia coli,E.coli)O121的O抗原基因簇的vio A基因的特异性序列设计引物和探针,通过对荧光聚合酶链式反应(real-time PCR)扩增条件的优化,建立检测E.coli O121的血清型特异性荧光PCR方法。E.coli O121标准菌株呈现扩增曲线,其它22株非O121 E.coli和19株非E.coli细菌的菌株均无扩增,检测的灵敏度可达155拷贝/反应。339份食品样品用EC肉汤增菌后用本荧光PCR法进行检测,检出E.coli O121阳性9份,阳性率为2.7%,其中市场上采购获得的生猪肉和生牛肉阳性率达4.1%。上述试验结果表明,本方法特异性强、操作简便,食品中E.coli O121检测全过程可在1.5 d内完成,适用于食品中E.coli O121的快速检测。

One set of primers and probe were used to amplify vioA gene of Escherichia coli O121 ,andaserogroup -specific real -time PCR method for detection of E. coli O121 was developed with optimization ofamplification conditions. PCR products of the reference strain showed a type amplification curve,and all of theother 22 stains of non-O121 E. coli and 19 stains of non-Escherichia coli bacteria didn＇t show any amplificationcurve. The detection limit of the real-time PCR assay was 155 copies per reaction mixture. 339 food sampleswere detected whether E. coli O121 present or not by the real time PCR method following enrichment in ECbroth,and9 samples were detected positive for this microorganism,namely with the positive ratio of 2.7%,inwhich the positive ratio of raw pork and raw beef samples purchased from markets was 4.1 %. The resultsuggested that the method developed was specific, easy to operate and 1.5d was enough for the whole process offood detection, and could be used for rapid detection of E. coli O121 in foods.