黄颡鱼小RNA病毒qPCR检测方法的建立与应用  

Establishment and Application of TaqMan⁃qPCR for Detection of Yellow Catfish Picornavirus

作　　者：穆雪姣 潘晓艺[2] 周可欣 蔺凌云[2] 姚嘉赟[2] 沈锦玉 MU Xuejiao;PAN Xiaoyi;ZHOU Kexing;LIN Lingyun;YAO Jiayun;SHEN Jinyu(Zhejiang Ocean University,Zhoushan 316000,China;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 313001,China)

机构地区：[1]浙江海洋大学,舟山316022 [2]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

出　　处：《病毒学报》2023年第4期1062-1071,共10页Chinese Journal of Virology

基　　金：湖州市乡村振兴项目(项目号:2020ZD2033),题目:黄颡鱼暴发病病原及其净化技术集成与示范。

摘　　要：黄颡鱼小RNA病毒(Yellow catfish Picornavirus,YCPrV)是黄颡鱼重要的致死性病原,本研究旨在建立一种快速、精准的YCPrV检测方法。基于YCPrV 22426⁃8毒株RdRp基因编码区序列设计特异性引物和探针,通过质粒标准品和标准曲线的制作,反应体系和反应条件的优化,灵敏度和特异性的测试,建立一种检测YCPrV的TaqMan荧光定量PCR(qPCR)方法。结果显示,在绘制的qPCR标准曲线中,拷贝数与Ct值具有良好的线性关系,相关系数(R^(2))为0.9974,扩增效率为88.98%,最高检测灵敏度为5.09×10^(0)拷贝。该方法对来自黄颡鱼、鲫鱼、鲤鱼和虾的其它8种病毒无扩增信号,仅YCPrV具有扩增曲线,对临床样品阳性检出率比普通PCR高12.28%。自然感染黄颡鱼组织中YCPrV检测结果发现,病毒载量从高到低依次为肠、肾、心、肝、脾、鳃和脑。以上结果表明,本研究建立的TaqMan荧光定量PCR检测方法具有良好的特异性、灵敏度和可重复性,临床检测YCPrV时,建议优选肾和心作为检测靶标组织。YCPrV qPCR方法的建立,可为YCPrV的精准定量检测和疾病早期预防提供有利手段。Yellow catfish Picornavirus(YCPrV)induces a serious and deadly viral disease that spreads rapidly in yellow catfish farms in China.To establish a rapid and accurate TaqMan⁃qPCR detection method for YCPrV,specific primers and a probe were designed based on the sequences of RdRp gene of YCPrV 22426⁃8 strain.Furthermore,the reaction system and conditions were optimized,and the plasmid standard and standard curve were established.The sensitivity and specificity of the method were tested.A TaqMan fluorescence quantitative RT⁃PCR method for detecting YCPrV was finally established.The results showed that the qPCR standard curve had a good linear relationship between copy number and Ct values,and with a correlation coefficient(R^(2))of 0.9974,and the amplification efficiency of 88.98%.The highest detection sensitivity reached 5.09×10^(0) copies.The method showed no amplification signal for other eight viruses from yellow catfish,crucian carp,common carp and shrimp.The positive detection rate of clinical samples was 12.28%higher than that of ordinary RT⁃PCR.The established TaqMan⁃qPCR was used to detect the tissues of yellow catfish naturally infected with YCPrV,and it was found that the order of viral load in tissues from high to low was intestine,kidney,heart,liver,spleen,gills and brain.The results above suggest that the TaqMan⁃qPCR detection method established has good specificity,repeatability and sensitivity,and that the kidney and heart are preferred as target tissues for detection of YCPrV.The method can be applied for the early detection of YCPrV in yellow catfish and disease prevention.

关 键 词：黄颡鱼 小RNA病毒 病毒性出血病 TaqMan荧光定量PCR 

分 类 号：S943[农业科学—水产养殖]