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沙门菌和副溶血性弧菌双重荧光PCR快速检测
方法的建立
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许如苏1，林彩华1，陈其生1，林志雄2，杨奇志3，蔡颖1
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（1.汕头出入境检验检疫局，广东 汕头515031；2.广东出入境检验检疫局，广东 广州510623；
3.罗氏诊断产品有限公司广州办事处，广东 广州510095）
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摘要：
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根据沙门菌invA基因和副溶血性弧菌toxR基因的保守序列设计引物和探针，通过优化反应条件与参数，建立了可同时检测沙门菌和副溶血性弧菌的双重荧光PCR方法。结果显示，该方法敏感度高，特异性强，重复性好。对沙门菌和副溶血性弧菌的检测低限均低于每反应体系10CFU，经对29株非目标菌进行检测均呈阴性，而定量检测批内和批间的变异系数均小于2%；应用该方法检测人工染菌样品和实际样品，结果与SN标准方法的检测结果一致；应用该法可在8h内完成对样品中沙门菌和副溶血性弧菌的同步检验。 |
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关键词：
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沙门菌；副溶血性弧菌；双重荧光PCR |
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中图分类号：S 852.61 文献标识码：
A 文章编号：1673-4696(2008)06-0465-05 |
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Development of a dual real-time PCR assay for rapid simultaneous
detection of Salmonella spp. and Vibrio parahaemolyticus |
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XU Ru-su1,LIN Cai-hua1,CHEN
Qi-sheng1,LIN Zhi-xiong2,YANG Qi-zhi3,CAI
Ying1 |
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(1．Shantou Entry-Exit Inspection and Quarantine Bureau,Shantou 515031,China；
2.Guangdong Entry-Exit Inspection and Quarantine Bureau,Guangzhou 510623,China；
3.Guangzhou Representative Office,Roche Diagnostics Limited Company,Guangzhou 510095,China) |
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Abstract: A dual real-time PCR assay was developed for the simultaneous detection of Salmonella spp. and Vibrio parahaemolyticus using primers and probe designed for the conservative domain of invA gene of Salmonella spp. and toxR gene of V.parahaemolyticus.The method was highly reproducible,specific and sensitive for detection of Salmonella spp. and V.parahaemolyticus,which could detect simulta-neously less than 10CFU Salmonella spp. and V.parahaemolyticus in the same reaction system.In the specificity test,29 non-target reference and standard strains were negative.All the variation coefficients of intra-assay and inter-assay were less than 2% in the quantitative tests.The assay was applied to samples experimentally-contaminated with the two pathogenic bacteria and to aquatic products for entry and exit,and the results were consistent with those of SN standards.It took less than 8h to qualitatively or quantitatively detect Salmonella spp. and V.parahaemolyticus in samples simultaneously. |
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Key words: Salmonella spp. ；Vibrio parahaemolyticus；dual real-time PCR |
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