基于分子马达生物传感器技术的副溶血性弧菌分子分型方法的初步研究

发布时间：2018-06-25 17:55

本文选题：副溶血性弧菌 + 分子马达　；参考：《微生物学通报》2013年07期

【摘要】：【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。
[Abstract]:[objective] to establish a simple and rapid molecular typing method based on molecular motor technology, and to classify virulent and non-virulent Vibrio parahaemolyticus rapidly. [methods] A molecular motor was constructed with F0F1-ATPase as the core. Four probes were designed using virulence gene of Vibrio parahaemolyticus tdhtrh and species-specific gene tlhfthotoxin R as target genes. F0F1-ATPase molecular motor biosensor was constructed by connecting the probe with molecular motor by biotin-avidin system. Ten Vibrio parahaemolyticus isolates were classified and compared with PCR-electrophoretic gel imaging results. At the same time, the sensitivity and specificity of biosensors were studied. [results] 10 of the 10 strains were tdh positive and 0 were trh positive, while all 10 strains were carrying tlh and toxr, which were consistent with the results of PCR-electrophoresis gel imaging. The minimum detection limit of molecular motor biosensor is 1 pg/ reaction system. The detection limit of PCR- electrophoresis gel imaging method for vibrio parahaemolyticus was 10 PG / PCR reaction system. [conclusion] Molecular typing method based on molecular motor was established. The detection sensitivity of Vibrio parahaemolyticus was 10 times higher than that of PCR- electrophoretic gel imaging, and the specificity was very high. The method is simple, rapid, time-saving and labor-saving. It is suitable for monitoring Vibrio parahaemolyticus and tracing the epidemiology of Vibrio parahaemolyticus in the basic laboratories of local disease control department and port quarantine department.
【作者单位】：北京出入境检验检疫局;威海出入境检验检疫局;中国合格评定国家认可中心;中国科学院生物物理研究所;
【基金】：国家质检总局科技计划项目(No.2011IK191,2012IK179)
【分类号】：R378

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