副溶血性弧菌LUX^TM荧光PCR快速检测方法的建立

Development of a LUX^(TM) PCR assay for rapid detection of Vibrio parahaemolyticus

根据LUXTM荧光PCR技术原理,针对副溶血性弧菌toxR基因的保守序列,设计了特异的LUXTM荧光标记引物,通过优化反应条件与参数,建立了可快速检测副溶血性弧菌的LUXTM荧光PCR方法。结果显示,该方法高度敏感,对纯菌的检测低限达到每个反应体系7个菌(CFU),经6 h增菌培养后检测,对样品的检测低限(CFU)达到100/25 g;特异性强,对副溶血性弧菌2株标准菌株和10株参考菌株的检验均呈阳性,而对霍乱弧菌等28株非目标菌的检测均呈阴性;重复性好,定量检测的批内和批间变异系数均小于1%。应用此方法检测水产样品103份,检出阳性样品6份,与TaqMan荧光PCR和SN标准的检测结果完全一致。用此方法可在8 h内完成对样品中副溶血性弧菌的检测,其检测的快速性、敏感性和特异性与TaqMan荧光PCR技术相当,但检测成本更低。

A LUX^TM PCR method was developed for rapid detection of Vibrio parahaemolyticus based on the primers designed for the conserved domain of toxR gene of V. parahaemolyticus by optimizing reaction conditions and parameters. The results showed that the method possessed high sensibility for detection of V. parahaemolyticus,which could detect 7 bacteria（CFU） per PCR reaction and 10^0/25 g after 6 h-culture of contaminated samples. In the specificity test,2 standard and 10 reference strains of V. parahaemolyticus were all positive,and 28 reference/standard strains of other bacteria such as Vibrio cholera were all negative. All of the coefficient of variation of intra-assay and inter-assay were less than 1% in the quantitative tests. The method was applied to detect 103 samples of aquatic products,6 of which were positive. The results were consistent with those of TaqMan PCR and SN. It took less than 8 h to detect V. parahaemolyticus. LUXTM PCR was not different from TaqMan PCR in rapidity, specificity and sensitivity and it was cheaper than TaqMan PCR.