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目的:建立双重荧光PCR对单增李斯特菌(LMO)特异性检测同时检测其毒力基因的方法。方法:根据LMO溶血素基因hlyA和內华素基因InlA的保守序列分别设计特异性引物和Taqman探针,优化多重荧光PCR反应体系,进行特异性、敏感性试验。结果:用建立的方法检测LMO标准菌株和24株分离株均出现hlyA和InlA扩增曲线,而沙门菌等其他菌株未见扩增曲线;敏感性试验结果方法的敏感性达到2.5×102 cfu/ml。结论:本研究建立的LMO实时荧光PCR检测方法特异性好、灵敏度高,是快速检测LMO及其毒力基因的有效手段,可用于食品中LMO检测。
Objective: To establish a dual fluorescent PCR method for the detection of virulence genes of Listeria monocytogenes (LMO). Methods: Specific primers and Taqman probes were designed according to the conserved sequence of hlyA gene of LMO hemolysin and InlA of endothelin gene respectively, and the multiplex fluorescence PCR reaction system was optimized for specificity and sensitivity test. Results: The amplification curves of hlyA and InlA were detected by the established method in LMO standard strains and 24 isolates, but no amplification curve was found in other strains such as Salmonella. The sensitivities of the sensitivity test results to 2.5 × 102 cfu / ml. Conclusion: The LMO real-time PCR detection method established in this study has good specificity and high sensitivity, which is an effective method for rapid detection of LMO and its virulence genes. It can be used for LMO detection in food.