三种食源性致病菌多重PCR检测方法的建立及初步应用

目的建立快速检测沙门菌、变形杆菌和金黄色葡萄球菌的多重聚合酶链反应（PCR）方法。方法本研究依据沙门菌侵袭基因正调节蛋白（hilA）基因、变形杆菌溶血素（hpmA）基因和金黄色葡萄球菌特异性pSa-442序列，运用PrimerPremier5．0分别设计3对特异性引物，预计PCR扩增的目的基因片段分别为为580bp、401bp、256bp。通过对单管多重PCR扩增的特异性、敏感性分析以及建立L16（4^3）正交试验对单管多重PCR扩增条件如引物浓度、dNTP浓度和Tm值等的优化，建立了快速同时检测3种食源性致病菌的单管多重PCR方法。结果该方法检测的灵敏度分别为：94．07pg沙门菌DNA，140．85ng变形杆菌DNA，1．41ng金黄色葡萄球菌DNA。模拟检测食品中的混合3种菌，4h培养后样品的最低检测限度分别为：沙门菌10^0菌落形成单位（CFU）／mL、变形杆菌10^1CFU／mL、金黄色葡萄球菌10^0CFU／mL。结论该方法特异性和灵敏度高，检验周期短，可用于对食品中多种致病菌的快速诊检和监控。

Establishment and application of multiplex PCR for diagnosing three food-borne bacterial pathogens

Objective To establish a muhiplex polymerase chain reaction （PCR） assay for simultaneous detection of Salmonella spp. , Proteus mirabilis and Staphylococcus aureus. Methods Three pairs of primers have been designed according to the hilA of Salmonella spp. , hemolysin （hpmA） of Proteus mirabilis and Staphylococcus aureus clone pSa- 442 Sau3AI fragment and the anticipated PCR products were 580bp, 401bp and 256bp. A multiplex PCR method has been developed for dignosing Salmonella spp. , Proteus mirabilis and Staphylococcus aureus, after analysis for specificity and sensitivity and optimization of reaction condition by orthogonal experimental design L16（4^3 ）. Results The sensi- tivity of multiplex PCR was 94.07 pg genome DNA for Salmonella spp. , 140.85 ng for Proteus mirabilis and 1.41 ng for Staphylococcus aureus. Simulation experiment showed that the minimum detection limit after cultivation for 4 hours was 10^0 CFU/ml for Salmonella spp. , 10^1 CFU/m] for Proteus mirabilis and 10^0 CFU/ml for Staphylococcus aureus. Conclusions A rapid, specific and sensitive multiplex PCR system has been established and it is valuable for exploration and appliacation.