多重PCR检测霍乱弧菌的毒素相关基因

Detection of the Virulence-associated Genes in Vibrio Cholerae by Multiplex PCR Assay

目的探索建立一种快速、敏感地检测霍乱弧菌O1群、O139群、非O1/非O139群的毒素相关基因的方法。方法针对霍乱弧菌霍乱肠毒素A亚单位基因(ctxA)、小带联结毒素基因(zot)、辅助霍乱肠毒素基因(ace)、霍乱弧菌毒素共调菌毛A基因(tcpA)、毒素表达调控蛋白基因(toxR)分别设计引物,建立多基因PCR方法;通过一次扩增反应之产物经琼脂糖凝胶电泳,可检测出菌株的毒素相关基因的携带情况。结果阳性对照菌株MO45(霍乱弧菌O139群)检出5种毒素相关基因,结果符合设计要求;其他不同来源的霍乱弧菌(O1群、O139群、非O1/非O139群)检出1～5种不等的毒素相关基因;根据毒素相关基因携带情况,可将菌株分为5个基因型,并可区分为产毒株和非产毒株;多重PCR敏感度可达102cfu/ml。结论该方法快速、特异、敏感,具有较大的应用价值。

Objective To study a rapid and sensitive method for determination of virulence-associated genes in O1El Tor,O139,non-O1/non-O139Vibrio cholerae strains.Methods Five pairs of primers were designed respectively ac-cording to cholera toxin sub-unit A gene(ctxA),accessory cholera enterotoxin gene(ace),zonula occludens toxin gene(zot),toxin coregulated pilus A gene(tcpA)and toxR regulatory protein gene(toxR).Multiplex PCR(MPCR)procedures for simultaneously detecting those five genes were established.The gene information of the virulence-associated genes in the Vibrio cholerae strains was obtained through agar gel electrophoresis for products of single amplification of the MPCR.Results The five virulence-associated genes in the positive control Vibrio cholerae O139(MO45strain)could be detected and the results were correct,which could meet the designed request for the method.In the other tested strains(O1EL Tor,O139,non-O1/non-O139)could be detected1to5kinds of the virulence-associated genes.Based on the results of the variety of carried virulence-associated genes,the tested Vibrio cholerae strains could be classified as5kinds of genetypes,and the Vibrio cholerae could be distinguished between toxic and non-toxic strains.The sensitivity of the MPCR approach reached to10 2 cfu/ml.Conclusion This method is rapid,specific and sensitive,which possess great value for practical application.