单增李斯特菌毒力因子多重荧光PCR法 建立与应用

Detection of Listeria monocytogenes in food using multiplex real-time PCR targeting the toxic genes

中文关键词:  多重荧光PCR  单增李斯特菌  hlyA  inA  actA

英文关键词:multiplex real-time PCR  Listeria monocytogenes  hlyA  inA  actA

中文摘要:

      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hlyA、内化素基因inlA和表面蛋白actA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410 cfu/mL; 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inlA、actA、hlyA 3种毒力基因, 另2株为毒力基因actA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。

英文摘要:

      Objective To establish a multiplex real-time PCR method for the determination of Listeria monocytogenes and 3 toxic genes, and apply for the detection of food. Methods The specific primers and probes were designed in the conserved region of the hlyA gene, inlA gene and actA gene. The reaction condi-tions were optimized, and the sensitivity, specificity, and stability of the assay were evaluated. Results The results showed high specificity for the detection of L. monocytogenes without any evident cross-reaction with other pathogens such as E.coli, S.aureus, S.typhi, C.freumdii, P.mirabillis and E.tarda. The detection limit was 410 cfu/mL in pure culture. It demonstrated the high reproducibility with a coefficient of variation (CV) less than 2%. Investigation of 84 samples showed that the real-time PCR method was fully compatible with the standard method and 4 positive results were detected. The multiplex real-time PCR method provided results more rapidly and can be performed in 2 working days compared to up to 7 d for the standard method. Two isolates have 3 toxic genes of hlyA, inlA and actA, and the other two were lack of actA gene. The results suggested that the pandemic strains of L. monocytogenes currently did not come from the same source. Conclusion The method has the potential to be used as an alternative to the standard method for food quality assurance providing rapid detection of L. monocytogenes in food. It has a high sensitivity and specificity, and provides fast and reliable method for the detection of foodborne diseases.