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当前位置: 首页 > 文章 > TaqMan探针双重荧光PCR法检测副溶血性弧菌 食品科学 2013,34 (18) 263-266
Position: Home > Articles > Detection of Vibrio parahaemolyticus Using Duplex Fluorescence Real-Time PCR with TaqMan Probe FOOD SCIENCE 2013,34 (18) 263-266

TaqMan探针双重荧光PCR法检测副溶血性弧菌

作  者：
许龙岩;袁慕云;曹际娟;凌莉
单  位：
广东出入境检验检疫局;辽宁出入境检验检疫局
关键词：
副溶血性弧菌;toxR基因;tdh基因;TaqMan探针;双重荧光聚合酶链式反应;检测;食品
摘  要：
目的:建立同时检测副溶血性弧菌(VP)和其毒力基因的方法。方法:根据VP的toxR基因和tdh基因,分别设计引物和探针,建立基于TaqMan探针双重荧光聚合酶链式反应扩增体系,进行特异性、敏感性实验,并用建立的方法检测从广东地区和辽宁地区进出口食品中分离的VP菌株的毒力基因分布情况。结果:VP标准菌株和3株从食物中毒病人中分离株均出现toxR基因和tdh基因扩增曲线,而31株包括溶藻弧菌、单增李斯特菌等弧菌属和肠杆菌科的菌株扩增结果呈阴性;敏感性实验结果表明,VP浓度与Ct值有很好的反向的线性关系,toxR和tdh线性方程的R2分别为0.999、0.997,最低检测浓度达到3.6×102CFU/mL;检测食品中分离的37株VP只出现toxR基因扩增曲线,未见tdh基因扩增曲线,表明37株VP分离株均未带tdh毒力基因。结论:建立的方法特异性好、灵敏度高,可用于食品中VP的特异性及毒力基因检测。
译  名：
Detection of Vibrio parahaemolyticus Using Duplex Fluorescence Real-Time PCR with TaqMan Probe
作  者：
XU Long-yan;YUAN Mu-yun;CAOJi-juan;LING Li;Guangdong Entry-Exit Inspection and Quarantine Bureau;Liaoning Entry-Exit Inspection and Quarantine Bureau;
关键词：
Vibrio parahaemolyticus;;toxR gene;;tdh gene;;TaqMan based probe;;duplex fluorescence real-time PCR;;detection
摘  要：
Objective: To establish a specific method for the detection of Vibrio parahaemolyticus(VP) and its virulence gene.Methods: Based on the sequences of toxR(transmembrane transcription activator) and tdh(thermostable direct hemolysin) genes from VP,specific primers and TaqMan probes were designed and a duplex fluorenscencet real-time PCR amplification system was established.The specificity and sensitivity of this method were evaluated.Moreover,the distribution of virulence gene in import and export foods from Guangdong and Liaoning provinces was investigated by this method.Results: The specificity tests showed that both toxR and tdh genes were amplified through the DNAs of standard strain,ATCC33847,and 3 wild strains isolated from food-poisoning patients.However,no specific amplification curves were observed for 31 other strains tested,including Vibrio alginolyticus and L.monocytogenes both from the genus Vibrio and Enterobacteriaceae.The sensitivity results demonstrated an inverse linear relationship between VP concentration and Ct values.The linear coefficients(R2) for toxR and tdh were 0.999 and 0.997 with an identical LOD of 3.6 × 102CFU/mL,respectively.Furthermore,the detection results revealed that toxR gene of all 37 foodborne VP wild strains in this study were positive for toxR gene,but all of them were negative for tdh gene,indicating that the genomes of 37 wild strains contained toxR gene,but did not contain tdh virulence gene.Conclusion: A rapid,sensitive and specific method suitable for the simultaneous detection of species and virulences specific genes of VP in foods has been established in this study.

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