基于LAMP技术针对溶藻弧菌gyrB基因快速检测方法的建立

Establishment of the Rapid Detection Method Targeting to GyrB Gene of Vibrio Parahaemolyticus Based on LAMP Technology

目的应用环介导等温扩增技术(Loop-mediated isothermal amplification, LAMP)建立针对溶藻弧菌gyrB基因的快速检测方法。方法以溶藻弧菌标准株(ATCC-17749)和溶藻弧菌野生株(WT)为研究对象,通过在线生物学软件(http://primerexplorer.jp/e/)设计针对溶藻弧菌gyrB基因的LAMP引物,利用恒温水浴进行等温扩增,优化反应条件,建立快速检测方法。结果①经2g/dl凝胶电泳结果显示反应温度为61℃时扩增产物成像效果较60℃,62℃和63℃明显,为适宜反应温度;②在61℃条件下反应60 min和90 min凝胶电泳成像效果差异不大,反应时间为60 min能满足扩增需要;③利用同一体系对临床6种其它常见致病菌进行等温扩增时未出现阳性条带,提示整个体系特异度较好;④采用模板稀释法验证该LAMP体系检测的灵敏度为10-4mg/L。结论建立了基于LAMP技术针对溶藻弧菌gyrB基因的快速检测方法,具有良好的特异度和敏感度,对快速检测溶藻弧菌有重要意义。

Objective To establish a rapid method for the detection of Vibrio alginolyticus by loop mediated isothermal amplification(LAMP). Methods Taking the Vibrio alginolyticus(ATCC-17749)and Vibrio alginolyticus(WT)as the research object, designing of LAMP primers for the gyrB gene by online biology software(http://primerexplorer.jp/e/),isothermal amplification was carried out in a constant temperature water bath, and the reaction conditions were optimized, and established a quick test response system. Results ① The results of 2 g/dl gel electrophoresis showed that the imaging effect of the amplified products was obviously higher than that of 60℃, 62℃ and 63℃ at the reaction temperature of 61℃. ② There was no significant difference in gel electrophoresis effect at 60 min and 90 min at 61℃, and the reaction time was 60 min, which could meet the need of amplification. ③ There was no positive band in isothermal amplification of 6 other common clinical pathogenic bacteria using the same system, suggesting that the specificity of the whole system was good. ④ Template dilution method was used to verify the sensitivity of the LAMP system for detection of 10-4 mg/L. Conlusion A rapid detection method for gyrB gene of Vibrio alginolyticus based on LAMP technology was established, which has good specificity and sensitivity, and has important significance for rapid detection of Vibrio alginolyticus.