十二种血清型蓝舌病病毒型特异性实时荧光定量RT⁃PCR定型方法的建立  

Establishment of Serotype ⁃ specific Real ⁃ time qRT ⁃ PCR Method for 12Serotypes of the Bluetongue Virus

作　　者：李占鸿 杨振兴[1] 宋子昂 李华春[1] 李卓然 廖德芳[1] 杨恒[1] LI Zhanhong;YANG Zhenxing;SONG Ziang;LI Huachun;LI Zhuoran;LIAO Defang;YANG Heng(Yunnan Animal Science and Veterinary Institute,Yunnan Tropical and Subtropical Animal Virology Laboratory,Kunming 650224,China;College of Veterinary Medicine,Yunnan Agricultural University,Kunming 650201,China)

机构地区：[1]云南省畜牧兽医科学院云南省热带亚热带动物病毒病重点实验室,昆明650224 [2]云南农业大学,动物医学院,昆明650201

出　　处：《病毒学报》2021年第5期1158-1166,共9页Chinese Journal of Virology

基　　金：国家自然科学基金(项目号:31760744)题目:蓝舌病病毒新血清型毒株感染与基因重配特性的研究;国家重点研发计划(项目号:2017YFC1200505),题目:蓝舌病等虫媒性动物疫病现场快速检测与实验室鉴定技术研究;公益性行业(农业)科研专项(项目号:201303035),题目:重要牛羊虫媒病毒病防控关键技术研究与应用;云南省中青年学术和技术带头人后备人才培养项目(项目号:2017HB055)。

摘　　要：为建立蓝舌病病毒(Bluetongue virus,BTV)十二种血清型(血清6、8、10、11、13、14、17、18、19、20、22与23型)特异性实时荧光定量RT⁃PCR(qRT⁃PCR)检测方法,根据GenBank公布的十二种BTV血清型毒株的基因节段2序列,选择高度保守区域,设计每种BTV血清型的扩增引物与TaqMan探针;以十二种血清型BTV参考毒株的cDNA为模板,进行引物的筛选,建立BTV血清型特异性qRT⁃PCR检测方法;对检测方法的灵敏度、特异性与重复性进行验证,分别以含有50、100与200个BTV噬斑形成单位(Plaque forming unit,PFU)的模拟BTV阳性血液样本为检测对象,进行检测效果的评估。实验结果表明,建立的BTV血清型特异性qRT⁃PCR检测方法具有良好的灵敏度和特异性,对不同血清型BTV核酸拷贝数的检出下限在12拷贝/μL(BTV⁃8)至57拷贝/μL(BTV⁃14)之间;对我国反刍动物上广泛流行的流行性出血病病毒、中山病病毒与阿卡斑病毒核酸的检测结果均为阴性。反应具有良好的重复性,组内Ct值的变异系数在0.92%至1.96%之间,组间Ct值的变异系数在0.26%至1.62%之间。对模拟BTV阳性血液样本的检测结果显示,BTV血清型特异性qRT⁃PCR可有效检测含50个PFU(噬斑形成单位)的BTV血液样本。本研究建立的十二种BTV血清型特异性qRT⁃PCR定型方法具有特异性强、灵敏度高和耗时少等优点,可用于动物感染BTV血清型的诊断。To establish serotype-specific real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)for detection of 12 serotypes bluetongue virus(BTV). Serotype-specific amplification primers and TaqMan^(TM) probes for each of BTV serotype were designed based on the highly conserved regions of the segment 2(Seg-2)sequences of BTV serotype 6,8,10,11,13,14,17,18,19,20,22 and 23 strains published in GenBank. qRT-PCR method for typing 12 serotypes of BTV was established using cDNA as templates,synthesized from homologous BTV reference strains,to screen primers. Specificity,sensitivity and repeatability of the qRT-PCR method were verified and the practical performance of the method was also evaluated using simulated BTV infection blood samples contained 50,100 and 200 plaque forming units(PFU)of BTV. Our results showed that the established BTV serotype-specific qRT-PCR methods possessed high sensitivity,strong specificity and good repeatability:lower limits for viral nucleic acids detection ranged from 12 copies/μL(BTV-8)to 57 copies/μL(BTV-14)without cross-reaction with nucleic acids of Epidemic hemorrhagic disease virus(EHDV),Chuzan virus(CHUV)and or Akabane virus(AKAV),which widely prevalent in ruminants of China;and the intra-and inter-group coefficient of variation(CV)were varying from 0.92%-1.96% and 0.26%-1.62%,respectively. The detection results of simulated BTV infection blood samples showed that BTV serotype specific qRT-PCR could effectively detect blood samples containing 50 PFU of BTV. In summary,The BTV serotype-specific qRT-PCR detection methods established in this study were specific,sensitive and time-saving,which could be applied for the rapid and accurate typing BTV infected animals.

关 键 词：蓝舌病病毒(BTV) 血清型 实时荧光定量RT⁃PCR 检测方法 

分 类 号：S852.65[农业科学—基础兽医学]