外源性绵羊肺腺瘤病毒实时荧光RPA检测方法的建立  

Establishment of real-time fluorescent RPA detection method for exogenous jaagsiekte sheep retrovirus

作　　者：刘然 张琳 刘淑英[1,2,3] LIU Ran;ZHANG Lin;LIU Shuying(College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot 010018,China;Key Laboratory of Clinical Diagnosis and Treatment of Animal Diseases,Ministry of Agriculture,Hohhot 01o018,China;Key Laboratory of Basic Veterinary Medicine,Hohhot 010018,China)

机构地区：[1]内蒙古农业大学兽医学院,内蒙古呼和浩特010018 [2]农业农村部动物临床诊疗技术重点实验室,内蒙古呼和浩特010018 [3]内蒙古自治区基础兽医学重点实验室,内蒙古呼和浩特010018

出　　处：《中国兽医学报》2023年第2期278-284,共7页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金面上资助项目(32072819);内蒙古科技重大专项计划资助项目(2021ZD0010);内蒙古应用研究资助项目(2019GG240);内蒙古草原英才创新团队资助项目(20151031)。

摘　　要：为实现对外源性绵羊肺腺瘤病毒(exJSRV)的快速检测,根据基因组中序列保守的env基因设计特异性引物和探针,通过优化反应条件和反应体系,建立了exJSRV的实时荧光RPA检测方法,并通过灵敏度、特异性、重复性及样品检测进行评价。结果显示,该方法最佳反应温度为40℃,用时短,仅需20 min即可完成扩增;灵敏度高,最低可检测到10^(6)ng/L的cDNA模板,纯化后的cDNA模板最低可检测到10 ng/L,与PCR结果一致;特异性强,能准确检测到exJSRV,与小反刍兽疫病毒(PPRV)、羊败血性链球菌(OSS)、牛肠道病毒(BEV)、传染性鼻气管炎病毒(IBRV)、牛副流感病毒3型(BPIV3)、肺炎支原体(MO)等继发呼吸道症状的病原无交叉反应;重复性好,对于相同质量浓度的模板cDNA检测结果一致;可有效检测出病羊肺组织、鼻液及外周血白细胞内的exJSRV,与PCR结果一致。结果表明,该方法可实现exJSRV的快速检测,为绵羊肺腺瘤病(OPA)的诊断及防控提供可靠依据。In order to realize the rapid detection of exogenous jaagsiekte sheep retrovirus(exJSRV),specific primers and probes were designed according to the conserved env gene in the genome.By optimizing the reaction conditions and reaction system,a real-time fluorescent RPA detection method for exJSRV was established,and its sensitivity,specificity and repeatability were evaluated and sample detection was conducted.The results showed that the best reaction temperature of this method was 40℃,and it took only 20 min to complete the amplification;it had high sensitivity,the lowest cDNA template could be detected at 10^(6) ng/L,and the purified cDNA template could be detected at least 10 ng/L,which was consistent with the PCR results;it had strong specificity,it could accurately detect exJSRV,and had no cross-reactivity with peste des petits ruminant virus(PPRV),ovine Streptococcus septicum(OSS),bovine enterovirus(BEV),infectious rhinotracheitis virus(IBRV),bovine parainfluenza virus type 3(BPIV3),mycoplasma pneumonia(MP)and other pathogens secondary to respiratory symptoms;it had good reproducibility,consistent detection results for template cDNA of the same mass concentration was obtained;it could effectively detect exJSRV in lung tissue,nasal fluid and peripheral blood leukocytes of sick sheep,which was consistent with the PCR results.In summary,this method can realize the rapid detection of exJSRV and provide a reliable basis for the diagnosis,prevention and control of ovine pulmonary adenoma(OPA).

关 键 词：绵羊肺腺瘤 绵羊肺腺瘤病毒 重组酶聚合酶恒温扩增技术 

分 类 号：S852.65[农业科学—基础兽医学]