副溶血性弧菌MPCR-DHPLC检测方法的建立  

Development of a multiplex PCR-DHPLC method for detecting Vibrio Parahaemolyticus

作　　者：凌莉[1] 许龙岩[1] 袁慕云[1] 胡科锋[1] 陈碧玲[1] 焦红[1] 

机构地区：[1]广东检验检疫技术中心,广州510623

出　　处：《中国卫生检验杂志》2013年第4期793-796,共4页Chinese Journal of Health Laboratory Technology

基　　金：广东省科技计划项目(2009B060600013)

摘　　要：目的:建立多重PCR(Multiplex PCR,MPCR)结合变性高效液相色谱(Denaturing High-Performance Liquid Chromatography,DHPLC)检测副溶血性弧菌的方法,并了解该菌携带毒力基因的情况。方法:根据副溶血性弧菌的毒力基因tdh、trh和种特异性基因toxR进行引物设计,以其单一PCR扩增产物在DHPLC出现的色谱峰作为标准色谱峰,建立MPCR-DHPLC的检测方法。结果:该方法能有效扩增副溶血性弧菌的tdh、trh和toxR基因,25株副溶血性弧菌的MPCR扩增产物经DHPLC分析所得的色谱峰在位置和峰型与标准色谱峰有很好的符合性,其他菌属的菌株均无特异性扩增,对模拟污染样品可正确检测,灵敏度达到1*103CFU/ml,特异性达到100%。结论:本文建立的MPCR-DHPLC方法可准确检测副溶血性弧菌,并了解该菌携带毒力基因的情况。Objective：To develop a multiplex PCR-DHPLC method for detecting Vibrio Parahaemolyticus and understand the virulence genes carried by the Vibrio Parahaemolyticus.Methods： According to the virulence genes tdh,trh from Vibrio Parahaemolyticus and species-specificity genes toxR,the primer was designed,the chromatographic peak of single PCR amplification products from DHPLC was used as standard peak and the detection method of Vibrio Parahaemolyticus was established.Results： The method could detect genes tdh,trh and toxR from Vibrio Parahaemolyticus.The chromatographic peak of multiplex PCR amplification products of 25strains of Vibrio Parahaemolyticus detected by DHPLC was conformed to the standard peak.The amplification products of other strains showed no specificity.The method could detect imitative contamination samples correctly,the sensitivity was up to 1×103 CFU/ml and the specificity was 100%.Conclusion： A multiplex PCR-DHPLC method established in the paper could detect Vibrio Parahaemolyticus and understand the virulence genes carried by the Vibrio Parahaemolyticus.

关 键 词：副溶血性弧菌 MPCR DHPLC 检测 

分 类 号：R378.3[医药卫生—病原生物学]