鳗鲡疱疹病毒SYBR GreenⅠ实时荧光定量PCR检测方法的建立与应用  

Establishment and application of SYBR GreenⅠreal-time fluorescence quantitative PCR for detection of Anguillid herpesvirus

作　　者：李英英[1] 杨金先[1] 陈曦[1] 陈强[1] 宋铁英[1] 葛均青[1] LI Yingying;YANG Jinxian;CHEN Xi;CHEN Qiang;SONG Tieying;GE Junqing(Institute of Biotechnology,Fujian Academy of Agricultural Sciences,Fuzhou 350003,China)

机构地区：[1]福建省农业科学院生物技术研究所,福建福州350003

出　　处：《水产学报》2021年第5期769-777,共9页Journal of Fisheries of China

基　　金：国家自然科学基金(31101933);福建省公益类科研院所基本科研专项(2017R1019-5);福建省农业科学院项目(AC2017-1)。

摘　　要：为了建立鳗鲡疱疹病毒(Anguillid herpesvirus,AngHV)的SYBR GreenⅠ实时荧光定量PCR(qPCR)检测方法,利用PCR扩增出AngHV ORF49的序列,克隆至pET-32a载体,构建重组质粒pET-32a-ORF49,作为qPCR的标准品;根据ORF49序列设计引物,以梯度稀释的质粒标准品为模板,进行SYBR GreenⅠqPCR扩增,制作标准曲线,建立AngHV的qPCR检测方法,并评价其灵敏性、特异性、重复性和应用效果。结果显示,qPCR的阈值循环数(cycle threshold value,CT)与标准品的拷贝数线性关系良好,且线性范围广,获得的标准曲线相关系数(R^(2))达到0.999,扩增效率为100.855%;该方法特异性好,可特异性检测AngHV,而对鲤疱疹病毒Ⅲ型(Cyprinid herpesvirus 3,CyHV-3)、蛙虹彩病毒(Rana grylio virus,RGV)和鳗鲡虹彩病毒(Eel iridovirus,EIV)无扩增;该方法重复性强,组内和组间变异系数分别小于1%和2%;该方法灵敏性高于普通PCR法,最低可检测到10个病毒拷贝,而普通PCR法为1000个病毒拷贝。qPCR检测发现,在感染AngHV的欧洲鳗鲡主要组织内均可检测到AngHV,并且鳃、鳍和皮肤黏液内的病毒量显著高于其他组织;对保存的25份疑似鳗鲡"脱黏败血综合征"病料进行检测,阳性检出率为96%,而普通PCR法的阳性检出率仅为76%。本研究建立的AngHV SYBR GreenⅠqPCR检测方法灵敏性高、特异性强、重复性好,可用于AngHV的快速和定量检测,对鳗鲡"脱黏败血综合征"的防控也具有重要意义。China owns the largest eel culture industry in the world.Since 1990 s,"mucus sloughing and hemorrhagic septicemia disease"has become one of the main epidemic diseases of cultured juvenile eel,with typical symptoms of"mucus sloughing","red head"and"hemorrhagic septicemia",and high morbidity and mortality.The disease could also be found in adult eels with typical symptoms of"hemorrhagic septicemia",resulting in a huge number of deaths,and enormous economic losses to the industry.Cultivation experience showed that the disease might be caused by viral pathogen.We had Anguillid herpesvirus 1(AngHV)isolated from the clinical samples of the disease.Further pathogenicity and epidemiological analysis showed that AngHV was the pathogenic agent of eel"mucus sloughing and hemorrhagic septicemia disease".A rapid and sensitive detection method for AngHV is of great significance for the early diagnosis,prevention and control of AngHV disease.Real-time fluorescence quantitative PCR(qPCR)method has been widely used in pathogen detection.To establish SYBR GreenⅠqPCR method for detection of AngHV,the sequence of AngHV ORF49 was amplified by PCR and cloned into pET-32 a vector to construct the standard plasmid pET-32 a-ORF49.Primers were designed according to ORF49 sequence,and a SYBR GreenⅠqPCR method was developed for AngHV detection using serially diluted standard plasmid as templates,then the sensitivity,repeatability,specificity and application effects of the method were evaluated.The results showed that the cycle threshold value(CT)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 0.999,and the amplification efficiency was 100.855%.The melting curve analysis showed that there was only one melting peak with Tm values of 88.34-88.95°C.Amplication specificity analysis indicated that the method could specifically detect AngHV,and had no amplification of Cyprinid herpesvirus 3(CyHV-3),Rana grylio virus(RGV)and Eel i

关 键 词：鳗鲡疱疹病毒 荧光定量PCR SYBR GreenⅠ 检测方法 

分 类 号：S941.41[农业科学—水产养殖]