多重PCR检测三种重要食源性致病菌方法的建立及应用

doi:10.3864/j.issn.0578-1752.2011.11.016

Scientia Agricultura Sinica ›› 2011, Vol. 44 ›› Issue (11): 2334-2340 .doi: 10.3864/j.issn.0578-1752.2011.11.016

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

WANG Hui, ZHU Rui-liang, TAN Yan-ling, WEI Kai, WANG Xin-jian, SUN Zhen-hong, SHENG Peng-cheng

山东农业大学动物科技学院/山东省动物生物工程与疾病控制实验室

Received:2010-10-08 Revised:2010-12-23 Online:2011-06-01 Published:2011-06-01

Abstract

Abstract:

【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。

Key words: Proteus mirabilis, Salmonella, Listeria monocytogenes, multiple PCR assay

WANG Hui,ZHU Rui-liang,TAN Yan-ling,WEI Kai,WANG Xin-jian,SUN Zhen-hong,SHENG Peng-cheng. Establishment and Application of Multiple PCR for Diagnosing Proteus mirabilis, Salmonella and Listeria monocytogenes[J].Scientia Agricultura Sinica, 2011, 44(11): 2334-2340 .

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