鳗鲡圆环病毒SYBR Green Ⅰ qPCR检测方法的建立与应用  

Establishment and application of SYBR Green I real-time fluorescence quantitative PCR for detection of Eel circovirus

作　　者：林而舒 LIN Er-shu(Freshwater Fisheries Research Institute of Fujian Province,Fuzhou 350000,China)

机构地区：[1]福建省淡水水产研究所,福州350000

出　　处：《淡水渔业》2023年第3期54-61,共8页Freshwater Fisheries

基　　金：福建省公益类科研院所基本科研专项(2022R11010014-5,2023R11010017-4)。

摘　　要：为建立鳗鲡圆环病毒(Eel circovirus,EeCV)的SYBR GreenⅠ实时荧光定量PCR(qPCR)检测方法,根据EeCV复制相关蛋白(replication-associated protein,RAP)基因的保守序列设计特异性引物,并评价其灵敏性、特异性、重复性和应用效果。qPCR的阈值循环数(Cycle threshold value,C t值)与标准品的拷贝数线性关系良好,且线性范围广(1.0×108~102拷贝数/μL);可特异性检测EeCV,而对鳗鲡疱疹病毒(Anguillid herpesvirus)、猪圆环病毒(Porcine circovirus)、鸭圆环病毒(Duck circovirus)无扩增;重复性强,组内和组间变异系数均小于3%;灵敏性高于普通PCR法10倍。且qPCR检测后发现,在感染EeCV的鳗鲡体内重要器官均可检测到EeCV,其中鳃的病毒量显著高于其他组织;对保存的44份疑似鳗鲡病毒性感染病料进行检测,阳性检出率为98%,而普通PCR法的阳性检出率为73%。China owns the largest eel culture industry in the world.Eels were rich in nutrition and delicious in meat and very popular among consumers.However,with the development of intensive and high-density eel breeding,there are more and more diseases,and the harm is becoming increasingly severe.Among them,viral eel disease had the characteristics of acute onset,short disease course and high mortality rate,which caused significant losses to eel farmers.Eel circovirus(EeCV)was one of the most common viruses in the eel breeding process.Establishing a sensitive and rapid EeCV detection method was important for early diagnosing and controlling eel virus infectious diseases.Real-time fluorescence PCR(qPCR)method has been widely used in pathogen detection.The sequence of EeCV replication-associated protein was amplified by PCR and cloned into pUCm-T to construct the standard plasmid pUCm-T-RAP and establish SYBR GreenⅠqPCR method for detection of EeCV.Primers were designed according to EeCV replication-associated protein,and an SYBR GreenⅠqPCR method was developled for EeCV detection using serially diluted standard plasmid as templates.The sensitivity,repeatability,specificity and application effects of the method were evaluated.The results showed that the cycle threshold(C t)of the qPCR assay had a good linear relationship with the copy number of the standard plasmid.The correlation coefficient(R 2)of the obtained standard curve was 0.9951.Amplication specificity analysis results indicated that the method could specifically detect EeCV,and had no amplification of Anguillid herpesvirus,Porcine circovirus,Duck circovirus.The coefficient of variation within and between groups was less than 3%,which indicated that the method was repeatable.The qPCR method could detect a minimum of 100 viral copies/μL with high sensitivity than the conventional PCR method,which has a detection limit of 1000μL.Application analysis indicated that EeCV could be detected in the primary tissues of EeCV infected Anguilla anguilla with 1.51×107 c

关 键 词：鳗鲡圆环病毒(Eel circovirus) 荧光定量PCR SYBR GreenⅠ 检测方法 

分 类 号：S943[农业科学—水产养殖]