罗氏沼虾野田村病毒和十足目虹彩病毒1二重荧光定量PCR检测方法的建立及应用  

Development and application of duplex fluorescent quantitative PCR method for detecting Macrobrachium rosenbergii nodavirus and decapod iridescent virus 1

作　　者：黄玉英 杨明伟 谭红连 梁正 曾兰 韦信贤 黄光华 童桂香 HUANG Yu-ying;YANG Ming-wei;TAN Hong-lian;LIANG Zheng;ZENG Lan;WEI Xin-xian;HUANG Guang-hua;TONG Gui-xiang(Guangxi Academy of Fishery Sciences/Guangxi Key Laboratory of Aquatic Genetic Breeding and Healthy Aquaculture,Nanning,Guangxi 530021,China;Guangxi Aquatic and Animal Husbandry School,Nanning,Guangxi 530021,China)

机构地区：[1]广西水产科学研究院/广西水产遗传育种与健康养殖重点实验室,广西南宁530021 [2]广西水产畜牧学校,广西南宁530021

出　　处：《南方农业学报》2022年第5期1474-1482,共9页Journal of Southern Agriculture

基　　金：广西重点研发计划项目(桂科AB1850016,桂科AB19245033,桂科AB21076008);广西农业科技自筹经费项目(Z202065)。

摘　　要：【目的】建立能同时检测罗氏沼虾野田村病毒(MrNV)和十足目虹彩病毒1(DIV1)的二重荧光定量PCR,为罗氏沼虾白尾病(WTD)及虹彩病毒病(DIV1D)的临床诊断和疫情监测提供更简便和高效的检测方法。【方法】分别根据MrNV-CP基因和DIV1-MCP基因的保守序列设计特异性引物和TaqMan-MGB探针,将目的基因片段分别克隆至pGM-T载体上制备重组质粒(pGM-T-CP^(MrNV)和pGM-T-MCP^(DIV1)),其中,pGM-T-CP^(MrNV)用Sal I酶切后经体外转录获得MrNV标准品RNA,pGM-T-MCP^(DIV1)则直接作为DIV1标准品DNA;以标准品为模板进行反应条件优化及标准曲线制定,建立可同时检测MrNV和DIV1的二重荧光定量PCR,并对该方法进行特异性、敏感性、重复性及临床应用试验。【结果】制备的MrNV标准品RNA和DIV1标准品DNA均具有很好的稳定性,可作为标准品和阳性对照使用;标准品起始模板范围为1.0×10^(1)~1.0×10^(8)Copies/反应时,建立的标准曲线具有良好线性关系。优化后的二重荧光定量PCR灵敏度高,对标准品的检测灵敏度可达10 Copies/反应,对罗氏沼虾组织样品的检测灵敏度约10 Copies/mg;与其他虾类常见病原无交叉反应,且整个检测过程仅需1 h左右。二重荧光定量PCR检测MrNV的组内试验和组间试验变异系数分别为0.31%~0.93%和0.96%~1.33%,检测DIV1的组内试验和组间试验变异系数分别为0.35%~0.81%和0.78%~1.04%。应用建立的二重荧光定量PCR和国内现行有效标准的套式PCR分别对117份临床样品进行MrNV和DIV1检测,结果显示对MrNV和DIV1检测的符合率分别为99.15%和97.44%,且二重荧光定量PCR检测目标病毒拷贝数较低样品时较套式PCR具有更高的灵敏度。2020年广西罗氏沼虾样品MrNV和DIV1的阳性检出率分别为6.0%和11.0%。【结论】结合TaqMan-MGB探针技术建立的二重荧光定量PCR能同时检测MrNV和DIV1,且具有灵敏度高、特异性强、重复性好、简便快速的特点,可为WTD和DIV1D的【Objective】To develop duplex fluorescent quantitative PCR detection method for Macrobrachium rosen-bergii nodavirus(MrNV)and decapod iridescent virus(DIV1),so as to provide a simpler and more effective detection technology for clinical diagnosis and epidemic monitoring of M.rosenbergii white tail disease(WTD)and decapod irides-cent virus 1 disease(DIV1D).【Method】Specific primers and TaqMan-Minor Groove Binder(TaqMan-MGB)probes were designed based on the conserved sequence of MrNV-CP gene and DIV1-MCP gene respectively,and the target gene frag-ments were cloned into the pGM-T carrier to construct recombinant plasmids,pGM-T-CP^(MrNV)and pGM-T-MCP^(DIV1).Among them,pGM-T-CP^(MrNV)was digested by SalⅠand transcribed in vitro to obtain MrNV standard RNA,while DNA of pGM T-MCP^(DIV1)were served as DIV1 standard DNA.The reaction conditions were optimized with standard being taken as tem-plates.And the standard curves were established.Then the duplex fluorescent quantitative PCR detection method for MrNV and DIV1 was developed,and the specificity,sensitivity,reproducibility tests and detection of clinical samples were car-ried out to evaluate the applicability of this method.【Result】The prepared RNA transcribed and DNAhad good stability and could meet the requirements of the standards,and the standard curves showed a good linear relationship with quantita-tive range from 1×10^(1)to 1×10^(8)copies/reaction.The optimized duplex fluorescent quantitative PCR method had a high sen-sitivity with the detection limit as low as to 10 copies/reaction for the standards and approximately 10 copies/mg for the M.rosenbergii tissue samples.It had no cross reaction with common shrimp pathogens,and the entire detection could be completed within 1 h for a single sample;the variation coefficients of intra-group and inter-group were 0.31%-0.93%and 0.96%-1.33%for MrNV detection,which were 0.35%-0.81%and 0.78%-1.04%for DIV1 detection.The duplex fluores-cent quantitative PCR method and nested PCR method recommended by current s

关 键 词：罗氏沼虾野田村病毒(MrNV) 十足目虹彩病毒1(DIV1) TAQMAN-MGB探针 二重荧光定量PCR 

分 类 号：S945.41[农业科学—水产养殖]