新型恒温核酸扩增法快速检测副溶血性弧菌的研究

Rapid Detection of Vibrio parahaemolyticus by Loop-mediated Isothermal Amplification Method

利用DNA环介导恒温核酸扩增法(loop-mediated isothermal amplification of DNA,LAMP)设计一对外引物和一对内引物,通过引物特异性识别tlh基因上的六个独立区域来快速检测副溶血弧菌。同时将检测结果与PCR方法进行比较。结果表明,LAMP反应在65℃恒温条件60min内完成,凝胶电泳呈现梯型条带;肉眼观察阳性结果出现白色混浊现象,添加1×SYBRGreenI荧光染料后,绿色的阳性结果很明显区别于橙色阴性结果;LAMP方法的最低检出限为10CFU/ml,PCR方法为103CFU/ml,LAMP方法检测灵敏度是PCR方法的100倍。因此,LAMP方法用于快速检测副溶血弧菌具有检测过程简单、反应结果肉眼即辨别并且灵敏度高特异性强的特点。实验装置简便,能够提供稳定热源即可,所以LAMP方法特别适合于现场快速诊断。

A pair of outer primers and a pair of inner primers were specially designed for recognizing six distinct sequences on target tlh gene by loop-mediated isothermal amplification of DNA （LAMP） method. The sensitivities of LAMP method and PCR were conpared for specific detection of Vibrio parahaemolyticus. The results showed that target DNA was amplified and visualized ladder-like pattern of bands on agarose gel by the detection system within 60 min at isothermal temperature 65 ℃; White precipitate allows easy and rapid detection of target DNA by naked eyes; The resulting amplicons are visualized by adding SYBR Green Ⅰ to the reaction tube; The LAMP assay has a detection limit of 10 CFU/ml, while that of PCR is 103 CFU/ml, that is to say that the sensitivity of LAMP is 100 times of that of PCR. So the LAMP method reported here demonstrates a potential and valuable means for detection of Vibrio parahaemolyticus especially for its rapidity, simplicity and low cost. The only equipment needed for the amplification reaction is a regular laboratory water bath or heat block that furnishes a constant temperature. The LAMP assay is particularly suitable for rapid clinical diagnosis.