中国兽医科学2025，Vol.55Issue(5)：695-704,10.DOI:10.16656/j.issn.1673-4696.2025.0074
绵羊肺腺瘤病毒和梅迪-维斯纳病毒双重实时荧光RPA检测方法的建立
Establishment of double real-time fluorescence RPA for detection of JSRV and MVV
摘要
Abstract
To develop a real-time fluorescence recombinase polymerase amplification(RPA)assay for simultaneous detection of jaagsiekte sheep retrovirus(JSRV)and maedi-visna virus(MVV),Specific primers and probes were designed and selected based on the conserved regions within the JSRV envelope gene and the MVV LTR sequence.The reaction system and conditions were optimized,and the assay was thor-oughly evaluated for its specificity,sensitivity,and reproducibility.The assay was validated using laboratory-preserved lung tissues with confirmed co-infections of JSRV and MVV,and the results were compared with a dual PCR method to determine the concordance rate.Our results showed that the assay was able to amplify target fragments within 20 minutes at 42 ℃ and detect cDNAs from JSRV and MVV co-infected lung tissues with the lowest detection limits of 1 ng/μL and 10 pg/μL,respectively.The results were negative for peste des petits ruminants virus,ovine streptococcosis septicemia,bovine en-teroviruses,infectious bovine rhinotracheitis virus,bovine parainfluenza virus type 3,and Mycoplas-ma ovipneumoniae,which are associated with secondary respiratory diseases in ruminants.Repeatability tests were conducted with templates at concentrations of 10 ng/μL and 100 ng/μL,resulting in a coeffi-cient of variation within the group ranging from 3.6％to 9.3％.Application of this method to clinical samples stored in the laboratory showed a 100％concordance rate with the dual PCR detection method.In conclusion,a dual real-time fluorescence RPA assay for the detection of JSRV and MVV was successfully established.This method is simple,rapid,and reliable,and could be utilized for differential diagno-sis of JSRV and MVV,as well as for clinical detection of concurrent infections.关键词
绵羊肺腺瘤病毒/梅迪-维斯纳病毒/重组酶聚合酶恒温扩增技术/快速检测Key words
jaagsiekte sheep retrovirus/maedi-visna virus/RPA/rapid detection分类
畜牧业引用本文复制引用
李慧萍,刘然,张良,刘淑英..绵羊肺腺瘤病毒和梅迪-维斯纳病毒双重实时荧光RPA检测方法的建立[J].中国兽医科学,2025,55(5):695-704,10.基金项目
国家自然科学基金项目(32072819,32360863) （32072819,32360863）
内蒙古科技重大专项(2021ZD0010) （2021ZD0010）
内蒙古草原英才创新团队项目(20151031) （20151031）
牛羊疫病防控与发育工程创新团队项目(BR22-13-08) （BR22-13-08）
兽医基础与牛羊疫病防控技术研发创新团队项目(NMGIRT2412) （NMGIRT2412）
内蒙古自治区教育厅一流学科科研专项(YLXKZX-NND-012) （YLXKZX-NND-012）
