| 摘要: |
| 采用分子生物学方法, 以霍乱弧菌lolB 为靶基因设计特异性引物, 进行了霍乱弧菌的PCR和环介导等温核酸扩增(Loop-mediated isothermal amplification, LAMP)检测技术研究, 并对它们的特异性、灵敏性和实际应用进行了比较。结果表明, 所建立的PCR 检测霍乱弧菌的方法最低检测限为4.0×103CFU/ml; LAMP检测方法在65℃下恒温扩增60min, 检测限为4.0×101CFU/ml, 反应产物加入荧光染料SYBR Green I后反应液呈现明显的绿色; 以温和气单胞菌、副溶血弧菌、鳗弧菌及美人鱼弧菌为对照菌株, 检测结果均为阴性; 霍乱弧菌人工染菌的8种水产品进行PCR及LAMP检测, 结果均为阳性, 而未染菌组均为阴性; PCR 及LAMP检测霍乱弧菌的方法均具有灵敏度较高、特异性强等优点, 且LAMP检测霍乱弧菌的方法灵敏度是PCR方法的100倍, 更适合于养殖现场检测的推广使用。 |
| 关键词: 霍乱弧菌, PCR, LAMP, 特异性, 灵敏性 |
| DOI：10.11693/hyhz201301031031 |
| 分类号: |
| 基金项目:江苏省水产三新工程重大项目资助, DY2012-3-7 号; 中央财政支持地方高校发展专项资金资助, CXTD16 号; 江苏高校优势学科建设工程项目资助, 2011 |
附件 |
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| COMPARISON BETWEEN PCR AND LAMP METHODS FOR THE DETECTION OF VIBRIO CHOLERAE ISOLATED FROM DISEASED MISGURNUS ANGUILLICAUDATUS |
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ZHANG Xiao-Jun, BAI Xue-Song, BI Ke-Ran, XU Jia-Tao, QIN Lei, YAN Bin-Lun
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College of Ocean, Key Laboratory of Oceanic Biotechnology of Jiangsu, Huaihai Institute of Technology
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| Abstract: |
| The specific primers of polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) were designed based on lolB gene. PCR and LAMP methods for the detection of Vibrio cholerae were established, and their specificity, sensitivity and practical application were compared in this study. The results showed the PCR primers could detect V. choleraeat the lowest level of 4.0×103 CFU/ml using PCR method, the LAMP primers could detect V. choleraeat the lowest level of 4.0×101 CFU/ml within 60min under isothermal condition at 65℃ using the LAMP detection system. The green amplified products were observed directly by naked-eye in the reaction tube by addition of SYBR Green I, and no cross reaction was detected in 4 kinds of control pathogenic bacteria including A. sobria,V. parahaemolyticus,V. anguillarum and V. damsela. The positive reaction was observed in PCR and LAMP detection system for artificial infected aquatic products, and control groups were negative. The studies revealed that they have equal effects between the PCR and LAMP in specificity and actual application, sensitivity level of LAMP is 100 times of the PCR method, and the LAMP method could be useful in the specific and rapid diagnose of the disease caused by V. cholerae in aquaculture. |
| Key words: Vibrio cholerae, PCR, LAMP, Specificity, Sensitivity |
