副溶血性弧菌PCR检测方法的建立

Detection of Vibrio parahaemolyticus by multiplex polymerase chain reaction

为了建立适于基层单位应用的针对副溶血性弧菌检测的特异PCR方法,检测副溶血性弧菌的基因序列。针对该菌的属特异性基因t1基因进行引物设计,扩增片段大小为449 bp。运用该PCR法可特异性的从副溶血性弧菌ATCC17802标准株中扩增出目的基因片段,与GenBank上发表的序列同源性为100%。而经常污染海产品的溶藻弧菌,嗜水气单胞菌标准株J-1,铜绿假单胞菌标准株ATCC27853结果均为阴性。该PCR法最低检出菌体DNA量为10-2 ng以及最低检出菌数为5.7×103 CFU/mL。对临床上送检的35份样品进行菌体DNA PCR方法检测并同时与国标GB/T4789.7-2003中使用的检测方法进行比对,两种检测方法的结果完全符合,与国标中使用的检测方法相比可大大缩短鉴定时间。因此该PCR法能快速鉴定当前副溶血性弧菌流行群,有利于流行病学溯源等研究。

To develop a PCR assay for detection of Vibrio parahaemolyticus, specific primers were designed based on t1 gene sequence of Vibrio parahaemolyticus, A PCR product of 449 bp was obtained, which was identical to the publicated sequence in GenBank. No PCR products were amplified from V.alginolyticus, A.hydrophila J-1 and P, aeruginosa ATCC27853. This method was capable of detecting 10^-2 ng DNA or 5.7×10^3 CFU/mL of Vibrio parahaemolyticus. Testing on 35 clinical samples showed that the PCR assay had the same detection rate as the standard method introduced in GB/T4789.7-2003. Therefore, the PCR assay provided a simple, fast and specific method for detection of Vibrio parahaemolyticus