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目的建立同时检测单增李斯特菌(Listeria monocytogenes)及其3种毒力因子的多重荧光PCR快速检测方法,并应用于日常食品的检测。方法根据单增李斯特菌溶血素基因hly A、内化素基因inl A和表面蛋白act A基因的保守序列,分别设计合成特异性引物和探针,优化多重荧光PCR反应体系。对该方法的特异性、敏感性和重复性进行评估。结果该法特异性强、敏感性高,对单增李斯特菌纯培养物的最低检出限410cfu/m L;重复性好,变异系数均小于2%。对84份食品检测结果与传统国标法相符,共检出单增李斯特菌4份,检出率为4.76%。多重荧光PCR检测方法耗时1 h,比传统方法节约2~5 d。4株单增李斯特菌分离株中2株同时含有inl A、act A、hly A 3种毒力基因,另2株为毒力基因act A缺失株,提示目前流行株并非同一来源。结论本研究建立的多重实时荧光PCR方法能同时对单增李斯特菌及其3种毒力因子进行快速检测,且灵敏度高、特异性好,为食源性疾病的病原学检测提供了快速可靠的方法。
OBJECTIVE To establish a rapid multiplex fluorescence PCR method for the simultaneous detection of Listeria monocytogenes and its three virulence factors and to apply it to routine food testing. Methods Specific primers and probes were designed and synthesized based on the sequences of hly A, Lysin, lysine, inl A, and act A genes. The multiplex PCR system was optimized. The specificity, sensitivity and reproducibility of the method were evaluated. Results The method was highly specific and sensitive. The detection limit of Listeria monocytogenes pure culture was 410 cfu / m L. The reproducibility was good and the coefficient of variation was less than 2%. The 84 food test results consistent with the traditional national standard method, a total of 4 Listeria monocytogenes were detected, the detection rate was 4.76%. Multiplex PCR detection method takes 1 h, than the traditional method of saving 2 ~ 5 d. Two isolates of four Listeria monocytogenes strains contained three virulence genes, inl A, act A and hly A, and the other two virulence genes act A deletion, suggesting that the current strains are not from the same source. Conclusion The multiplex real-time fluorescent PCR method established in this study can simultaneously detect Listeria monocytogenes and its three virulence factors rapidly and has high sensitivity and specificity and provides a fast and reliable method for the etiological detection of foodborne diseases Methods.