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| 水产动物致病性副溶血弧菌双重PCR 检测方法的研究 |
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张晓君1,2, 梁利国1,2, 阎斌伦1,2, 秦 蕾1,2, 戚耀之1,2
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1.淮海工学院海洋学院;2.江苏省海洋生物技术重点建设实验室
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| 摘要: |
| 副溶血弧菌(Vibrio parahaemolyticus)是多种水产养殖动物的主要病原菌, 尤其是可引起凡纳滨对虾幼虾呈毁灭性死亡。本研究基于gyrB 和toxR 两种基因序列设计2 对特异性引物, 建立一种副溶血弧菌快速、准确的双重PCR 检测方法, 扩增目的片段大小分别为285 bp 和368 bp。结果表明在同一PCR 反应体系中副溶血弧菌可同时扩增大小分别为285 bp 和368 bp 的2 种基因片段, 两种引物对4种其他水产动物病原菌无交叉反应; 敏感性检测结果显示, 该双重PCR 最低能检测8.867 2×103CFU/mL 菌体浓度的副溶血弧菌, 对副溶血弧菌模板DNA 的检出极限为0.029 3 mg/L; 对发病中国对虾糠虾幼体、水产品及虾池养殖用水进行双重PCR 检测, 呈阳性反应的样品可分离出优势生长的副溶血弧菌。该实验所建立的基于gyrB 和toxR 两种基因的双重PCR 检测方法可用于副溶血弧菌引起的水产动物疾病的快速诊断及分子流行病学的调查研究。 |
| 关键词: 副溶血弧菌(Vibrio parahaemolyticus) gyrB 基因 toxR 基因 双重PCR |
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| 基金项目:江苏省水产三项工程项目(PJ2010-58); 江苏省自然科学基金项目(BK2009163); 淮海工学院江苏省海洋生物技术重点建设实验室研究基金项目(2008HS016) |
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| Detection of pathogenic Vibrio parahaemolyticus isolated from aquatic animals by dulplex PCR |
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ZHANG Xiao-jun,LIANG Li-guo,YAN Bin-lun,QIN Lei,QI Yao-zhi
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| Abstract: |
| V. parahaemolyticus is a significant pathogen for the aquaculture industry, particularly devastating impact on cultivated Litopenaeus vannamei. In this study, two pair of specific primers was designed that allowed amplification of 285 bp and 368bp gene fragments based on gyrB and toxR genes. A simple dulplex polymerase chain reaction (PCR) that will detect V. parahaemolyticus were established. Consequently, two PCR primers could simultaneously amplify 285 bp and 368bp gene fragments from chromosomal DNA of V. parahaemolyticus in one PCR reaction, and no cross reaction was detected in 4 other pathogenic Vibrio species tested. The results of sensitivity of dulplex PCR showed that the two primers could detect V. parahaemolyticus at a level of as few as 8.8672×103CFU/mL, and detect purified chromosomal DNA at a level of as few as 0.0293 mg/L . Dominant V. parahaemolyticus could be isolated from the positive sample of dulplex PCR through detecting mysis, aquatic products and tank water samples. The PCR protocol amplifying gyrB and toxR gene fragments of the V. parahaemolyticus was established and could be useful in the specific and rapid diagnose of the disease caused by V. parahaemolyticus. |
| Key words: Vibrio parahaemolyticus gyrB gene toxR gene dulplex PCR |