流水式养殖动物中弧菌和嗜水气单胞菌的PCR法监测

PCR detection of pathogenic Vibrio and Aeromonas hydrophila in aquaculture animals in flowing-through systems

为了解流水式养殖场养殖动物中弧菌和气单胞菌的感染状况,分别针对霍乱弧菌肠毒素A亚单位(ctxA)基因、O139和O1群群特异基因、毒力协同调节菌毛A亚单位基因(tcpA)、中心调节蛋白基因(toxR)、副溶血弧菌不耐热溶血素基因(tlh)、创伤弧菌Vibrio vulnificus溶细胞素基因(vvhA)和嗜水气单胞菌的气溶素基因(aerA)设计引物,建立了聚合酶链式反应(PCR)检测技术,PCR产物经电泳,根据扩增条带的大小和数目,检测和区分霍乱弧菌、嗜水气单胞菌和副溶血弧菌。对南昌沿岸霍乱流行水域及附近养殖场的养殖动物进行了18个月的连续监测,对1 440份水产品的增菌液进行PCR检测,17份检出副溶血弧菌,25份检出霍乱弧菌,创伤弧菌和嗜水气单胞菌均未检出。扩增结果与传统培养检测法的结果一致。监测结果表明,南昌附近地区霍乱弧菌和副溶血弧菌检出率分别为1.74%和1.18%,总体感染状况处于较低水平,但个别养殖品种弧菌检出率较高。

To investigate the prevalence of pathogenic Vibrio and Aeromonas hydrophila in aquaculture animals in flowing through systems in Nanchang,animals were sampled over an 18-month period between March 2009 and September 2010.Seven pairs of primers were designed for toxin sub-unit A（ctxA） gene,O139 special gene,O1 special gene,tcpA gene,toxR gene,haemolytic gene,vvhA gene and aerolysin（aerA） gene.Specific PCR was established to simultaneously detect these 7 genes.Strains of Vibrio cholerae,V.parahaemolyticus,V.vulnificus and Aeromonas hydrophila can be detected and differentiated by the size and numbers of specific PCR products through electrophoresis bands examination.In 1 440 tested samples,17 samples were Vibrio parahaemolyticus positive（1.74%）,25 samples were Vibrio cholerae positive（1.18%）,and no Aeromonas hydrophila and Vibrio vulnificus were found in the samples.The results of PCR detection were consistent with those of the traditional culture methods.The results indicated that the levels of pathogenic Vibrio in Nanchang area were very low,while the levels of pathogenic Vibrio in Amyda Sincnsis and Rana catesbeiana were relatively high.