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【目的】建立基于分子马达技术的简便快速的分子分型方法,对携带和非携带毒力基因的副溶血性弧菌进行快速分类。【方法】以F0F1-ATPase为核心构建分子马达,以副溶血性弧菌毒力基因tdh、trh和种特异性基因tlh、toxR为靶基因设计4个探针。通过生物素-亲和素系统将探针与分子马达连接构建F0F1-ATPase分子马达生物传感器,对10株副溶血性弧菌分离株进行分类,并与PCR-电泳-凝胶成像结果进行比较;同时对生物传感器的检测灵敏度和特异性进行研究。【结果】10株试验菌株中10株tdh阳性,0株trh阳性,而10株菌都携带tlh和toxR,与PCR-电泳-凝胶成像结果一致;分子马达生物传感器的最低检测限为1 pg/反应体系,且能够对副溶血性弧菌特异性识别,PCR-电泳-凝胶成像方法的最低检测限为10 pg/PCR反应体系。【结论】建立了基于分子马达的分子分型方法,能够对副溶血性弧菌的致病性进行快速诊断,检测灵敏度比PCR-电泳-凝胶成像方法高了10倍,而且特异性非常高。该方法简便、快速、省时、省力,适用于地方疾控部门和口岸检疫部门的基层实验室开展副溶血性弧菌监测和流行病学溯源工作。
【Objective】 A simple and rapid molecular typing method based on molecular motor technology was established to rapidly classify Vibrio parahaemolyticus carrying and non-carrying virulence genes. 【Method】 The molecular motor was constructed with F0F1-ATPase as the core, and four probes targeting virulence genes tdh, trh and species-specific genes tlh and toxR were designed. The F0F1-ATPase molecular motor biosensor was constructed by biotin-avidin system probe and molecular motor to classify 10 isolates of Vibrio parahaemolyticus and compare them with PCR-gel electrophoresis. At the same time, the detection sensitivity and specificity of biosensors were studied. 【Results】 Ten strains were positive for tdh and 0 were positive for trh, while all ten strains carried tlh and toxR, which was consistent with PCR-gel electrophoresis. The detection limit of molecular motor biosensor was 1 pg / Reaction system and can specifically recognize Vibrio parahaemolyticus. The minimum detection limit of PCR-electrophoresis-gel imaging method is 10 pg / PCR reaction system. 【Conclusion】 A molecular typing method based on molecular motor was established to rapidly diagnose the pathogenicity of Vibrio parahaemolyticus. The detection sensitivity was 10 times higher than that of PCR-electrophoresis-gel imaging and the specificity was very high . The method is simple, rapid, time-saving and labor-saving, and is applicable to the local laboratories of primary disease control and quarantine departments of port authorities for monitoring and epidemiological traceability of Vibrio parahaemolyticus.