多重PCR检测三种重要食源性致病菌方法的建立及应用

Establishment and Application of Multiple PCR for Diagnosing Proteus mirabilis,Salmonella and Listeria monocytogenes

【目的】建立快速检测奇异变形杆菌、沙门氏菌和单核细胞增生李斯特氏杆菌3种食源性致病菌的多重PCR方法。【方法】根据奇异变形杆菌尿素酶合成的正向调节因子R基因(ureR)、沙门氏菌侵袭性抗原保守基因(invA)和单核细胞增生李斯特氏杆菌编码溶血素O(LLO)的hlyA基因,分别设计3对特异性引物,对单基因PCR和单管多重PCR扩增的特异性、敏感性分析以及建立L16(43)正交试验对单管多重PCR扩增条件,如引物浓度、Tm值、模板量等的优化,建立快速检测奇异变形杆菌、沙门氏菌和单核细胞增生李斯特氏杆菌的稳定的单管多重PCR方法。【结果】针对奇异变形杆菌、沙门氏菌和单核细胞增生李斯特氏杆菌3种食源性致病菌所设计的3对引物分别能扩增出374、724和215 bp的目的条带,具有高度特异性,反应条件优化后,3种目的菌在105 CFU/mL均可同时扩增出较清晰条带(奇异变形杆菌和单核细胞增生李斯特氏杆菌在104 CFU/mL浓度下仍然可见到条带),比单基因PCR低一个稀释度,并且人工模拟试验和市售食品试验检测结果稳定。【结论】该方法操作简单、快速、特异性强和灵敏度高,能够实现对奇异变形杆菌、沙门氏菌和单核细胞增生李斯特氏杆菌3种食源性致病菌的快速诊断检测和监控。

【Objective】 The aim of this study is to establish a multiplex polymerase chain reaction（PCR） assay for simultaneous identification of Proteus mirabilis,Salmonella and Listeria monocytogenes.【Method】Based on the gene sequences of positive adjustment factor R gene of Urease（ureR） in Proteus mirabilis,conservative invasive antigen gene（invA） in Salmonella and the hly gene in Listeria monoytogenes,a three pairs of primer were designed.The specificity and sensitivity of PCR were analyzed for single gene.Multiple PCR method has been developed after analysis and optimization reaction condition by orthogonal experimental design L16（43）.【Result】 Aiming to Proteus mirabilis,Salmonella and Listeria monocytogenes,under the optimized conditions,the anticipated PCR products were 374 bp,724 bp and 215 bp,respectively,and the specificity was high,and the minimum detection limit was 105 CFU/mL,which was a dilute degree lower in the sensitivity of single PCR.The results were stable in simulated examination and in marketing examination.【Conclusion】A rapid,specific and sensitive multiplex PCR system has been established and it is valuable for diagnosis and monitoring for Proteus mirabilis,Salmonella and Listeria monocytogenes.