水产学报2024，Vol.48Issue(4)：358-365,8.DOI:10.11964/jfc.20230714084
美洲鳗鲡腺瘤病毒(AEAdoV)普通PCR和SYBR Green Ⅰ实时荧光定量PCR检测方法的建立及应用
Establishment and application of routine PCR and SYBR Green Ⅰ real-time fluorescence quantitative PCR for detection of American eel adomavirus
摘要
Abstract
Haemorrhagic gill necrosis disease(HGND)has become one of the important epidemic diseases of cul-tured American eels.In previous studies,American eel adomavirus(AEAdoV)was isolated from eel with HGND.In order to establish a PCR and real-time fluorescence quantitative PCR(qPCR)method for the detection of the virus,primers were designed according to the superfamily 3 helicases(S3H)sequence of AEAdoV-FJ.Further evaluations on the sensitivity,specificity,repeatability,and application effect of this method were conducted.The results showed that the established PCR method could specially amplify a 300 bp band,which was cloned and used to construct plasmid standards for qPCR;the cycle threshold value(Ct)of qPCR and the copy number of the stand-ard sample had a consistent relationship with a wide range.The obtained correlation coefficient(R2)of the stand-ard curve reached 0.999,and the amplification efficiency was 105.067%.The qPCR method could detect a min-imum of 10 viral copies with higher sensitivity than routine PCR method.Both methods could specifically detect AEAdoV with negative amplification reaction on Rana grylio virus(RGV),Anguillid herpesvirus(AngHV),Koi herpesvirus(KHV),White spot syndrome virus(WSSV),Japanese eel adomavirus(JEAdoV),and Marbled eel adomavirus(MEAdoV).The coefficient of variation within and between groups of this method were both less than 2%,suggesting good repeatability.Further application results showed that the detection rate of AEAdoV from 35 samples of American eels with HGND by routine PCR and qPCR was 82.8%and 97%,respectively,indicating that the virus is prevalent among the diseased eels.Analysis of the virus quantity of the eel tissues indicated that the level of AEAdoV was relatively higher in heart,liver,gill and fin,and lower in mucus,skin and spleen.These results indicate that routine PCR and qPCR assays with high sensitivity and specificity for detection of AEAdoV have been established,and confirm that AEAdoV is closely related to HGND and exists in the main organs of infected eels.This study will be valuable for further investigations of the correlation between AEAdoV and HGND,and helpful for understanding the epidemic and etiology of AEAdoV.关键词
美洲鳗鲡腺瘤病毒(AEAdoV)/普通PCR/荧光定量PCR/SYBR GreenⅠ/检测方法Key words
American eel adomavirus(AEAdoV)/routine PCR/real-time fluorescence quantitative PCR/SYBR GreenⅠ/detection method分类
农业科技引用本文复制引用
孔文迪,陈曦,杨金先,葛均青..美洲鳗鲡腺瘤病毒(AEAdoV)普通PCR和SYBR Green Ⅰ实时荧光定量PCR检测方法的建立及应用[J].水产学报,2024,48(4):358-365,8.基金项目
福建省公益类科研院所基本科研专项(2021R1027007) （2021R1027007）
福建省农业科学院"5511"协同创新工程建设项目(XTCXGC2021013) Public-Interest Scientific Institution Basal Research Fund of Fujian Province(2021R1027007) （XTCXGC2021013）
"5511"Collaborative Innovation Project of Fujian Academy of Agricultural Sciences(XTCXGC2021013) （XTCXGC2021013）
