非洲马瘟病毒实时荧光定量RT-PCR检测方法的建立  

Development of Real-time Fluorescent Quantitative RT-PCR for Detection of AHSV Based on SYBR GreenⅠ

作　　者：赵文华[1] 杨仕标[1] 李富祥[1] 

机构地区：[1]云南省畜牧兽医科学院国家外来动物疫病诊断实验室云南省热带亚热带动物病毒病重点实验室,云南昆明650224

出　　处：《动物医学进展》2013年第12期11-16,共6页Progress In Veterinary Medicine

基　　金：公益性行业(农业)科研专项(200903037)

摘　　要：非洲马瘟病毒(AHSV)为双股RNA病毒,能感染所有马科动物。为建立一种AHSV快速检测方法,根据GenBank及作者所测AHSV 9个血清型VP7-ORF全长核苷酸序列,设计一对位于AHSV基因组S7片段的特异引物,然后进行荧光定量检测。经试验,证实该对引物对9种血清型的AHSV RNA均有特异性扩增,能检出特异荧光信号,而对与AHSV同属的蓝舌病病毒(BTV)、鹿流行性出血症病毒(EHDV)及正常马淋巴组织和阴性对照均无扩增,无有效荧光信号可检出。以国家外来动物疫病诊断实验室已构建好的AHSV 9型pMD18-T-VP7质粒为标准品可建立良好的标准曲线,实现对检测样本的实时定量。本研究建立了模板预变性结合"三步"法敏感特异的AHSV实时荧光定量RT-PCR快速检测方法,检测灵敏度可达102拷贝/μL。African horse sickness virus（AHSV）is a double-stranded ribonucleic acid（dsRNA）virus which can infect all the equid animals.Based on the VP7-ORF sequences from GenBank and results of sequencing for nine serotypes of AHSV previously completed by our own team,apair of specific primers located on the segment S7 were designed for SYBR GreenⅠquantitative detection,in order to develop one kind of quick method for detecting the AHSV.Results showed that the primer pair was identified to be specific for each of the 9 serotypes of AHSV only and specific fluorescent signal could be detected,but not for the related viruses including BTV,EHDV and normal lymphoid tissue of horse without specific fluorescent signal.The built positive recombinant plasmid（AHSV9pMD-18T-VP7）in our lab was used for a positive quantitative template to establish a standard curve.Finally,the method of pre-denaturalized template based on＂three-step＂reaction was established in the study.It was demonstrated that the SYBR GreenⅠfluorescent quantitative RT-PCR for detection of AHSV could be used for the rapid diagnosis and quantitative detection with detection limit of 102 copies/μL cDNA.

关 键 词：非洲马瘟病毒 血清型 SYBR Green Ⅰ 实时荧光定量RT-PCR 

分 类 号：S852.651[农业科学—基础兽医学] Q786[农业科学—兽医学]