蛙病毒3型类和桑蒂库帕蛙病毒类双重荧光定量PCR检测方法的建立与应用  

Establishment and application of a duplex real-rime PCR method for FV3-like virus group and Santee-Cooper-like ranavirus detection

作　　者：邓艳 柏建山 徐浩 季新成 黄燕琼 何淑华 曾伟伟 蒋茗 DENG Yan;BAI Jian-shan;XU Hao;JI Xin-cheng;HUANG Yan-qiong;HE Shu-hua;ZHEN Wei-wei;JIANG Ming(Ntegrated Technique&Service Center of Guangzhou Baiyun Airport Customs,Guangzhou 510460,China;Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Customs District,Shenzhen 518045,China;Center for International Inspection and Quarantine Standards and Technical Regulations,General Administration of Customs,Beijing 100013,China;Foshan Institute of Science and Technology Guangdong,School of Science and Engineering,Foshan 528225,China)

机构地区：[1]广州白云机场海关综合技术服务中心,广东广州510460 [2]深圳海关动植物检验检疫技术中心,广东深圳518045 [3]海关总署国际检验检疫标准与技术法规研究中心,北京100013 [4]佛山科学技术学院生命科学与工程学院,广东佛山528225

出　　处：《中国预防兽医学报》2025年第1期48-53,79,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：广州市科技项目(2023B04J0172);海关总署科研项目(2022HK006)。

摘　　要：为建立一种可快速鉴别检测蛙病毒3型(FV3)类和桑蒂库帕蛙病毒(SCRV)类的双重荧光定量PCR(qPCR)方法,本研究根据FV3类和SCRV类的主要衣壳蛋白(MCP)基因序列,设计一对特异性引物和两种不同荧光标记的探针,采用特异性引物扩增虎纹蛙病毒(TFV)和大口黑鲈虹彩病毒(LMBV)的目的基因,构建重组质粒标准品pMD19-T-TFV和pMD19-T-LMBV。采用矩阵法优化各反应条件后,初步建立了检测这两类蛙病毒的双重qPCR方法。标准曲线结果显示两个重组质粒标准品混合物与各自的Ct值均呈良好的线性关系。采用本研究建立的方法分别检测TFV、LMBV、伯勒虹彩病毒(BCV)、蛙病毒3型、流行性造血器官坏死病毒(EHNV)、锦鲤疱疹病毒、罗非鱼湖病毒、病毒性神经坏死病毒、斑点叉尾鮰病毒、鲤浮肿病毒、传染性造血器官坏死病毒、传染性脾肾坏死病毒、呼肠孤病毒Ⅱ型、疱疹病毒Ⅱ型,评估该方法的特异;以100~108稀释的重组质粒标准混合物为模板采用建立的双重qPCR方法检测,评估该方法的敏感性;以10^(2)~10^(8)稀释的重组质粒标准混合物为模板采用该双重qPCR方法分别进行组内和组间的重复性试验。结果显示,该方法仅能扩增出FV3类的TFV、BIV、FV3及EHNV和SCRV类的LMBV,其他病毒均无扩增曲线;对pMD19-T-TFV和pMD19-T-LMBV的检测限分别为1拷贝/µL和10拷贝/µL;组内变异系数和组间变异系数均小于2%。利用建立的双重qPCR方法和WOAH推荐的常规PCR方法分别对298份临床样品分别进行检测,结果显示,双重qPCR方法检出21份SCRV类,2份FV3类,普通PCR方法检出15份SCRV类,2份FV3类,双重PCR方法与常规PCR方法对FV3类和SCRV类的检测结果的阳性符合率均达100%。本研究建立的双重qPCR方法具有特异性强、敏感性高、重复性好等优点,对于多种蛙病毒属成员的鉴别检测和早期预警具有重要意义。In order to establish a duplex real-time PCR method for rapid and specific detection of FV3-like virus group and Santee-Cooper ranavirus,a pair of specific primers and two probes labeled with different fluorescent groups were designed based on the sequences of the major capsid protein genes.The target genes of Ambystoma tigrinum virus(ATV)and Largemouth bass ranavirus(LMBV)were amplified by specific primers,and recombinant standard plasmids pMD19-T-TFV and pMD19-T-LMBV were constructed.After optimizing the reaction conditions,the duplex TaqMan real-time PCR method was preliminarily established for simultaneous detection of these two viruses.The standard curve results showed a good linear relationship between the two recombinant plasmid standards and their respective Ct values.The method's specificity was evaluated by detecting TFV,LMBV,KHV,TiLV,VNNV,CCV,CEV,IHNV,ISKNV,GcRV-2,SCRV,CyHV-2;the sensitivity of the method was evaluated using a mixture of plasmids standard with dilutions ranging from 100 to 108;the repeatability of the method was evaluated using a mixture of plasmids standard with dilutions ranging from 102 to 108.The results showed that the method was positive only for FV3-like virus group and SCRV-like virus group and had no cross-reactivity with other tested viruses.The detection limits for pMD19-T-TFV and pMD19-T-LMBV were 1 copy/μL and 10 copies/μL respectively.The intra-assay and inter-assay coefficients of variation were less than 2%.298 clinical samples were tested by the established duplex real-time PCR method and the conventional PCR method recommended by WOAH.The results showed that 21 SCRV-positive samples and 2 FV3-like virus-positive samples were detected by the duplex real-time PCR method,while 15 SCRV-positive samples and 2 FV3-like virus-positive samples were detected by the conventional PCR method.The positive coincidence rates for FV3 and SCRV detections were 100%.In conclusion,the duplex real-time PCR method established in this study has the advantages of strong specificity,high se

关 键 词：蛙病毒 双重荧光定量PCR TAQMAN探针 

分 类 号：S947.2[农业科学—水产养殖]