基于 LAMP技术针对溶藻弧菌 gyrB基因快速检测方法的建立

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。

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

Objective　To establish a rapid method for the detection of Vibrio alginolyticus by loop mediated isothermalamplification （LAMP）. Methods　Taking the Vibrio alginolyticus（ATCC-17749）and Vibrio alginolyticus（WT）as theresearch 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, andestablished a quick test response system. Results　① The results of 2g/dl gel electrophoresis showed that the imaging effect ofthe amplified products was obviously higher than that of 60℃ , 62℃ and 63℃ at the reaction temperature of 61℃ . ② Therewas no significant difference in gel electrophoresis effect at 60 min and 90 min at 61℃ , and the reaction time was 60 min, whichcould meet the need of amplification. ③ There was no positive band in isothermal amplification of 6 other common clinicalpathogenic bacteria using the same system, suggesting that the specificity of the whole system was good. ④ Template dilutionmethod was used to verify the sensitivity of the LAMP system for detection of 10-4 mg/L. Conlusion　A rapid detection methodfor gyrB gene of Vibrio alginolyticus based on LAMP technology was established, which has good specificity and sensitivity, andhas important significance for rapid detection of Vibrio alginolyticus.