大口黑鲈弹状病毒qPCR检测方法的建立与临床应用  

Establishment and Clinical Application of qPCR Method for Detection of Micropterus salmoides Rhabdovirus

作　　者：陈雪兰 蔺凌云[1] 杨娜 王亿文 姚嘉赟[1] 沈锦玉[1,2] 潘晓艺[1] CHEN Xuelan;LIN Lingyun;YANG Na;WANG Yiwen;YAO Jiayun;SHEN Jinyu;PAN Xiaoyi(Ministry of Agriculture and Rural Areas Key Laboratory of Healthy Freshwater Aquaculture,Key Laboratory of Fish Health and Nutrition of Zhejiang Province,Zhejiang Institute of Freshwater Fisheries,Huzhou 3130l,China;School of Fisheries and Life,Shanghai Ocean University,Shanghai201306,China)

机构地区：[1]浙江省淡水水产研究所农业农村部淡水渔业健康养殖重点实验室浙江省鱼类健康与营养重点实验室,湖州313001 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《病毒学报》2024年第5期1033-1046,共14页Chinese Journal of Virology

基　　金：浙江省农业重大技术协同项目(项目号:2022XTTGSC01),题目:大口黑鲈养殖产业关键技术集成创新与示范;湖州市公益性应用研究项目(项目号:2021GZ24),题目:大口黑鲈弹状病毒减毒活疫苗的研制及初步应用;浙江省科技厅院所专项(项目号:2024YSZX02),题目:鳜鲈绿色养殖关键技术研究与示范。

摘　　要：大口黑鲈弹状病毒病是引起大口黑鲈幼苗暴发性死亡最严重的疾病,为满足大口黑鲈幼苗弹状病毒(Micropterus salmoides rhabdovirus,MSRV)的快速检测,建立大口黑鲈弹状病毒SYBR Green qPCR检测方法。根据大口黑鲈弹状病毒核蛋白基因的保守序列设计qPCR引物,对反应体系进行优化,制作标准曲线,并进行灵敏度、重复性和特异性测试,建立染料法qPCR检测方法,并在临床标本和人工感染鱼组织样品中进行应用。结果表明,构建的标准曲线,模板量与Ct值之间具有良好的线性关系,相关系数R2为0.9994,检测灵敏度达到4.5拷贝/μL,除MSRV、鳜弹状病毒(Sinipercachuatsirhabdovirus,SCRV)、杂交鳢弹状病毒(Hybridsnakeheadrhabdovirus,HSHRV)和斑鳢弹状病毒(Channamaculatarhabdovirus,CMRV)外,对其他7种病毒大口黑鲈蛙虹彩病毒(Largemouth bass ranavirus,LMBV)、传染性脾肾坏死病毒(Infectious spleen and kidney necrosis virus,ISKNV)、神经坏死病毒(Nervous necrosis virus,NNV),传染性造血器官坏死病毒(Infectious hematopoietic necrosis virus,IHNV)、鲤春病毒血症病毒(Spring viraemia of carp virus,SVCV)、草鱼呼肠孤病毒(Grass carp reovirus,GCRV)和黄颡鱼小RNA病毒(Yellow catfish picornavirus,YCPrV)都无扩增荧光信号;在对45份临床样品的检测中,建立的qPCR检出阳性率比套式PCR高11.11%;对人工感染MSRV的不同规格(1 g/ind、3 g/ind、8 g/ind)大口黑鲈各组织检测结果显示,脾脏中病毒载量最高,并且大规格鱼苗(8 g/ind)组织中病毒载量要低于小规格鱼苗(1 g/ind、3 g/ind),表明小规格鱼苗更易感。以上结果表明,建立的大口黑鲈弹状病毒SYBR Green qPCR检测方法是一种适合临床样品检测的特异性强、灵敏度高、稳定性好的MSRV定量方法。该方法的建立为MSRV疾病检疫提供了可靠的技术手段。Micropterus salmoides rhabdovirus disease is the most serious disease that causes sudden death of Micropterus salmoides fry.A SYBR Green qPCR method was established for rapid detection of Micropterus salmoides rhabdovirus(MSRV).The qPCR primers were designed according to the conserved sequence of the nucleoprotein gene of MSRV;the reaction system was optimized,the standard curve was established,and the sensitivity,repeatability and specificity were tested.The SYBR Green qPCR detection method was established and applied in clinical specimens and tissue samples of artificially infected fish.The results showed that there was a good linear relationship between the constructed standard curve,the amount of template and Ct value.The correlation coefficient R2 was 0.9994.The detection sensitivity reached 4.5 copies/μL.There was no amplification fluorescence signal with Largemouth bass ranavirus(LMBV),Infectious spleen and kidney necrosis virus(ISKNV),Nervous necrosis virus(NNV),Infectious hematopoietic necrosis virus(IHNV),Spring viraemia of carp virus(SVCV),Grass carp reovirus(GCRV)and Yellow catfish picornavirus(YCPrV),except MSRV,Siniperca chuatsi rhabdovirus(SCRV),Hybrid snakehead rhabdovirus(HSHRV)and Channa maculata rhabdovirus(CMRV).In 45 clinical samples,the positive rate with qPCR was 11.11%higher than that with nested PCR.The results of tissue detection of Micropterus salmoides artificially infected with MSRV(1g/ind,3g/ind,8g/ind)showed that the viral load in spleen was the highest,and the viral load in larger-sized fish fry(8g/ind)was lower than that in smaller-sized fish fry(1g/ind,3g/ind),indicating that smaller-sized fish fry are more susceptible.These results indicate that the established SYBR Green qPCR method is a MSRV quantitative method suitable for clinical sample detection with strong specificity,high sensitivity and good repeatability.This method provides a reliable technical means for MSRV disease control and prevention.

关 键 词：大口黑鲈 弹状病毒 qPCR 病毒检测 

分 类 号：S943[农业科学—水产养殖]