免疫磁分离-荧光PCR应用在肉类单增李斯特氏菌的检测

Detection of Listeria monocytogenes in meat products by means of immuno-magnetic cell separation combined with fluorescence PCR technique

目的建立适用于肉类产品中单核增生李斯特氏菌的荧光PCR检测方法。方法根据单增李斯特氏菌溶血素基因hlyA,设计合成引物和荧光探针,结合免疫磁珠筛选,选择简便的DNA提取方法,建立适用于检测肉类制品中单增李斯特氏菌的荧光PCR方法。对该方法进行特异性、灵敏度及模拟样品检测效果的研究。结果对20株不同菌属的标准菌株和30株野生李斯特氏菌菌株,及混合菌液的检测,结果显示该方法具有良好的特异性。对染菌模拟样本的检测结果表明,经过24h增菌,该方法的检测低限为1cfu/g,整个流程只需27h。结论免疫磁分离荧光PCR方法为快速、简便和准确检测肉类制品中单增李斯氏菌提供了一个新的途径。

To develop a method to detect Listeria monocytogenes in meat products by immuno-magnetic cell separation combined with fluorescence PCR technique, one set of primers and fluorescent probe was designed according to the sequences of the listeriolysin O gene （hly） of L. monocytogenes, and a simple method of DNA extraction combined with immuno-magnetic cell separation was selected. It was demonstrated that this method of assay was proved to be 100% specific, as determined with 30 wild strains and 20 standard strains of listeria and 5 mixed baterial suspensions. The threshold of bacterial detection was as low as 1 CFU/g with a simulative sample incubation time of 24 hours ,and the whole course of detection only required 27 hours. It is evident that this method of assay can provide a new approach for the rapid and correct detection of L. monocytogenes in meat products.