创伤弧菌和哈氏弧菌双重PCR检测方法的建立及初步应用  

Establishment and Preliminary Application of the Duplex PCR to Detection of Vibrio vulnificus and Vibrio harveyi

作　　者：陈福艳[1,2] 程光平[1] 欧阳贤华 陈晓汉[2] 梁万文[2] 韦友传[1] 黄婷[2] 杨学明[2] 陈明[2] 王瑞[2] 李丽萍[2] 韦明利[2] 

机构地区：[1]广西大学,广西南宁53004 [2]广西水产研究所,广西水产遗传育种与健康养殖重点实验室,广西南宁530021 [3]广西芳草安桂生物科技有限公司,广西南宁530001

出　　处：《广东海洋大学学报》2013年第3期52-55,共4页Journal of Guangdong Ocean University

基　　金：广西重点实验室计划项目(12-A-03-01);广西科技创新推广扶持项目(桂渔牧科1204905;桂渔牧科1204902);广西区直属公益性专项资金项目(GXIF-2012-02;GXIF-2010-05)

摘　　要：根据创伤弧菌(Vibrio vulnificus)的溶细胞毒素基因序列和哈氏弧菌(Vibrio harveyi)的toxR基因序列,分别设计并合成两对特异性引物,通过PCR反应条件优化,测试两种菌的特异性和敏感性,建立双重PCR方法,同时快速检测V.vulnificus和V.harveyi。结果表明:纯培养V.vulnificus和V.harveyi的检测灵敏度分别是12 cfu/mL和18 cfu/mL,与无乳链球菌、海豚链球菌、副溶血弧菌及美人发光杆菌无交叉反应;此PCR检测方法具有良好的特异性、敏感性,具快速、高效等优点,对细菌V.vulnificus和V.harveyi诊断与防治具有较好的临床应用性。As Vibrio vulnificus and Vibrio harveyi mainly affects marine cultured animals,it is necessary to establish a rapid detection method.According to the cytolysin gene sequence of V.vulnificus and toxR gene sequence of V.harveyi,two pairs of primers were designed and synthesized.The diagnosis of duplex polymerase chain reaction（PCR） for rapid identification of V.vulnificus and V.harveyi was established by optimization of PCR conditions and by testing specificity and sensitiveness.The duplex PCR test results showed that two specific and amplified fragments were produced which sizes were 128 bp for V.vulnificus and 211 bp for V.harveyi,and no cross-reaction tests were done with Streptococcus agalactiae,Streptococcus iniae,Bibrio parahemolyticus,and Vibrio damsela.The lowest concentrations of 12 cfu/mL for V.vulnificus and 18 cfu /mL for V.harveyi could be detected.The duplex PCR would be a specific and sensitive method which was timesaving,laborsaving,quick and high-efficient for identification of duplex V.vulnificus and V.harveyi.

关 键 词：双重PCR 创伤弧菌 哈氏弧菌 

分 类 号：Q93-331[生物学—微生物学]