【摘要】 目的基于多重聚合酶链反应(PCR)建立一种快速方便的检测副溶血弧菌"大流行菌群"及其毒力基因的方法。方法结合副溶血弧菌"大流行菌群"的群特异鉴定方法 GS-PCR,与副溶血弧菌致病相关的耐热直接溶血素基因(tdh)和耐热直接溶血素相关溶血素基因(trh)序列设计引物,建立并优化多重PCR检测方法。用已知的大流行菌株和非大流行临床分离株,对其进行特异性、灵敏度等性能的评价。并对224株食品分离株和3株临床分离株副溶血弧菌进行了再鉴定。结果副溶血弧菌"大流行菌群"有群特异PCR标识基因和tdh扩增条带。而其他细菌扩增结果呈阴性。检测灵敏度可达到105CFU/ml。对实验室保存的224株食品分离株和3株临床分离株进行再鉴定表明,其中5株为副溶血弧菌"大流行菌群",其中3株为tdh阳性、trh阴性,2株tdh、trh均阴性。结论本研究所建立的多重PCR方法特异性好、灵敏度高。为副溶血弧菌"大流行菌群"及其毒力基因的快速大规模检测提供良好的技术支持。更多还原

【Abstract】 Objective To establish a multiplex PCR assay for the rapid detection of pandemic group of Vibrio parahaemolyticus and toxic genes based on GS-PCR. Methods Primers w ere designed based on the sequences of three genes,including one group-specific gene,toxRS,and tw o toxic genes,thermostable direct hemolysin( tdh) and TDH-related hemolysin( trh). The specificity and sensitivity of the assay w ere tested w ith reference strains of pandemic and non-pandemic V. parahaemolyticus strains,and re-identification of 224 food borne strains and 3 clinical isolates of V. parahaemolyticus w as conducted. Results The toxRS and tdh w ere successfully amplified from the pandemic clone of V. parahaemolyticus. tdh w ere amplified from some clinical isolates of V. parahaemolyticus,w hile no amplicon w as detected in the other bacteria.Detection limits of the assay w as 105 CFU / ml. Of the 224 food borne isolates and 3 clinical isolates,5 w ere identified as pandemic clone,3 w ere tdh positive and trh negative,tw o w ere tdh negative and trh negative. Conclusion The established multiplex PCR assay is specific and sensitive for the simultaneous detection of pandemic group of V. parahaemolyticus and related toxic genes,w hich could provide sufficient support for the large scale surveillance for pandemic group of V.parahaemolyticus.更多还原