蛳鱼诺卡氏菌SYBRGreenⅠ实时荧光定量PCR检测方法的建立与应用  

Development of a SYBR GreenⅠreal-time PCR assay for detection of Nocardia seriolae and its application

作　　者：王国良[1] 刘璐[1] 李思源[1] 

机构地区：[1]宁波大学应用海洋生物技术教育部重点实验室,浙江宁波315211

出　　处：《水产学报》2012年第4期509-513,共5页Journal of Fisheries of China

基　　金：长江学者和创新团队发展计划(IRT0734);农业部公益性行业科研专项(200903029);浙江省自然科学基金(Y3080244)

摘　　要：根据GenBank中蛳鱼诺卡氏菌16S-23S rRNA基因序列设计并合成一对特异性引物,经反应体系优化后建立了检测蛳鱼诺卡氏菌的SYBR GreenⅠ实时荧光定量PCR方法。结果显示,该方法线性关系良好,标准曲线的相关系数为0.998;溶解曲线分析显示产物为单一的特异峰;检测灵敏度可达10 6μg/μL的DNA含量,与嗜水气单胞菌、大肠杆菌、金黄色葡萄球菌、副溶血弧菌、麦氏弧菌不发生交叉反应,具有良好的特异性。应用建立的方法在蛳鱼诺卡氏菌病爆发时期,对16份鱼体组织、养殖水体、饲料等样品进行了检测,结果 7份为阳性,与细菌分离、培养检查结果 100%相符。既能检测发病鱼,又能检出未发病且已感染的病鱼,对病害的早期防控体现应用价值。结果表明,建立的实时荧光定量PCR方法具有特异、敏感、快速、定量等优点,可用于蛳鱼类致病鱼诺卡氏菌的快速检测。According to the 16S-23S rRNA gene sequences of Nocardia seriolae available in GenBank,a pair of primers was designed for establishing a SYBR Green I real-time fluorescence quantitative PCR method.It had a good linear relationship between initial templates and Ct values,and the correlation coefficient of the standard curve was 0.998.The sensitivity analysis showed that the developed SYBR GreenⅠreal-time PCR could detect 10？6 μg/μL DNA.The specificity assay showed that negative control and the other bacteria such as Aeromonas hydrophila、Escherichia coil、Staphylococcus aureus,could not be detected by this PCR.Through the epidemic seasons,16 fish tissue sample,4 water samples and 8 fish bait tissue samples were detected by the real-time PCR assay.Results showed that 7 out of those samples were positive,which had good agreement（100%） with bacteriological analysis by isolation and culture.It was able to diagnose the clinically diseased fishes,and to recognize the carrier of N.seriolae as well.It reflected application value on early prevention and control of disease.The results showed that the developed SYBR GreenⅠreal-time PCR assay had the advantages of specificity,sensitivity,rapidity and quantitativity,and was able to be applied to the clinical diagnosis of N.seriolae in fish.

关 键 词：蛳鱼诺卡氏菌 SYBR-GreenⅠ 实时荧光定量PCR 

分 类 号：S941.4[农业科学—水产养殖]