【摘要】 为建立羊口疮病毒(ORFV)快速准确的检测方法，并明确安徽省主要肉羊养殖区流行毒株的遗传演化特征，本研究构建了一种TaqMan实时荧光定量PCR方法，对采自安徽省重点养羊地区的303份临床样本进行检测；同时通过PCR扩增阳性样品中ORFV的全长VIR基因，并进行测序与遗传变异分析。结果显示，所建立的实时荧光定量PCR方法可在约30 min内完成检测，对ORFV基因组DNA的检测下限为4.28拷贝/μL,且与其他5种常见病毒核酸无交叉反应，批内和批间扩增变异系数(Cv)均低于1.33%。临床样本检测结果显示，ORFV阳性率为48.84%(148/303)。对阳性样本中的ORFV全长VIR基因进行常规PCR扩增，获得64条完整的VIR基因DNA序列(山羊源33条，绵羊源31条)。序列分析显示，64条样本毒株VIR基因DNA及其氨基酸序列的同源性分别为95.1%～100%和92.4%～100%。其中，33条山羊源毒株VIR基因DNA与氨基酸序列的同源性分别为95.1%～100%和92.4%～100%,其氨基酸突变位点主要位于Q24H、S101A、S124P和A154T等处。31条绵羊源毒株VIR基因DNA与氨基酸序列的同源性为99.5%～100.0%,其氨基酸突变位点主要分布于R14S、D17G、D47N、M97L、M103I、H122R、K135E、L140R、A154V和A169T等处。进化树分析显示，33条山羊源毒株(Group 1)与31条绵羊源毒株(Group 2)分别位于遗传距离较远的两大分支。其中，山羊源毒株与我国山西、台湾、云南、吉林、福建和陕西山羊参考毒株亲缘关系较近；绵羊源毒株则与新疆、河南、甘肃和广西绵羊参考毒株亲缘关系较近。本研究建立了一种可快速检测ORFV的TaqMan实时荧光定量PCR方法，并揭示安徽省山羊源与绵羊源ORFV毒株VIR基因具有不同的突变模式且存在显著的遗传差异，为该病的防控提供了科学依据。更多还原

【Abstract】 To rapidly and accurately detect the Orf virus(ORFV) and identify the genetic variation of the prevalent ORFV strains in Anhui Province,a TaqMan real-time fluorescence quantitative PCR method for detecting ORFV was developed and used to detect the 303 clinical samples collected from important meat sheep breeding cities in Anhui Province.The full-length VIR genes of ORFV in the positive samples were PCR amplified,and genetic variation was analysed after sequencing.The results showed that the detection time of the established real-time fluorescence quantitative PCR method was approximately 30 minutes.It detected as low as 4.28 copies/μL of ORFV genomic DNA and showed no cross-reaction with the nucleic acid samples of five common viruses.The intra-and inter-coefficient variation values were lower than 1.33 %.Detection of the clinical samples revealed a positive rate of ORFV of 48.84%(148/303).Sixty-four complete VIR gene DNA sequences(33 from goat sources and 31 from sheep sources) were amplified using conventional PCR and sequenced.Sequence analysis showed that the homology of nucleic acid and amino acid sequences of the VIR genes of 64 sample strains was 95.1 %-100 % and 92.4%-100 %,respectively.Among them,the homology of those of the VIR genes of 33 goat-derived strains was95.1 %-100% and 92.4%-100%,and the amino acid mutation sites were mainly located at Q24H,S101A,S124P,and A154T,etc.The homology of those of the VIR genes of 31 sheep-derived strains was 99.5 %-100.0 %,and the amino acid mutation sites were primarily distributed at R14S,D17G,D47N,M97L,M103I,H122R,K135E,L140R,A154V,and A169T,etc.Phylogenetic tree analysis revealed that the 33 goat-derived strains(Group 1) and the 31 sheep-derived strains(Group 2) were positioned in two major branches,characterized by a relatively long genetic distance.The goat-derived strains were closely related to the reference goat strains from Shanxi,Taiwan,Yunnan,Jilin,Fujian,and Shaanxi,while the sheep-derived strains were more closely related to the reference sheep strains from Xinjiang,Henan,Gansu,and Guangxi.In summary,we established a TaqMan real-time fluorescence quantitative PCR method for the rapid and accurate detection of ORFV,and revealed the different mutation patterns and a significant genetic difference among the goat-derived and sheep-derived strains,which laid a basis for prevention and control of this disease.更多还原