双重LAMP技术快速检测水产品中副溶血性弧菌和霍乱弧菌的方法学研究

Study on Simultaneous Detection of Vibrio Parahaemolyticus and Vibrio Cholerae in Aquatic Products by Loop-Mediated Isothermal Amplification Method

摘要:

建立环介导等温扩增技术(LAMP)同时快速检测水产品中副溶血性弧菌和霍乱弧菌的方法。针对副溶血性弧菌toxR和霍乱弧菌ompW基因设计特异性引物，优化反应条件，建立水产品中副溶血性弧菌和霍乱弧菌的检测方法，并同时应用双重LAMP技术和PCR技术对实验菌株进行副溶血性弧菌和霍乱弧菌检测，比较两种方法的特异性和灵敏度。LAMP的最佳反应温度为61 ℃，在此条件下，双重LAMP检测技术检测副溶血性弧菌和霍乱弧菌DNA的敏感度可达3.12 fg，且与其他常见的细菌株无交叉反应，特异性为100%；对模拟食品样品进行直接检测时检测限为50 cfu/mL；对60份水产样品进行检测时，6份样品出现LAMP及PCR阳性，而传统培养方法检测出4份阳性。实验结果表明所建立的双重LAMP技术在检测水产品中副溶血性弧菌和霍乱弧菌时灵敏度、特异性高，时间成本低，适合于水产品中副溶血性弧菌和霍乱弧菌的快速检测。

Abstract:

A loop-mediated isothermal amplification (LAMP) method was developed for simultaneous and rapid detection of Vibrio parahaemolyticus and Vibrio cholerae in aquatic products. Based on the nucleic acid sequences of toxR of V. parahaemolyticus and ompW of V. cholerae, a pair of primers was designed for LAMP. After optimizing the reaction conditions, the levels of V. parahaemolyticus and V. cholerae in aquatic products were estimated using LAMP and PCR, and the detection rate and efficiency of the two methods were compared. The optimal reaction temperature was found to be 61 ℃; at this temperature, the detection limit for the DNA templates of V. parahaemolyticus and V. cholerae was 3.1 fg, and no cross-reaction with other common bacteria was found, yielding a detection specificity of 100%. For direct detection of V. parahaemolyticus and V. cholerae in artificially contaminated food samples, the detection limit was found to be 50 cfu/mL. In addition, when 50 seafood samples were tested by LAMP and PCR, 6 of them were found to be positive, but only 4 were detected to be positive by conventional microbiological methods. Thus, the LAMP method had high specificity and sensitivity for detecting V. parahaemolyticus and V. cholerae in aquatic products, and would be suitable for rapidly detecting V. parahaemolyticus and V. cholerae.