圆圈病毒人源1型实时荧光RAA快速检测方法的建立  

Establishment of Real-time RAA Detection Method for Gyrovirus Homsa1

作　　者：余丹 赵俊柯 尹文巧 张艳芳 李小凤 吴嫒琼 谢志勤 喻华英 谢芝勋 YU Dan;ZHAO Junke;YIN Wenqiao;ZHANG Yanfang;LI Xiaofeng;WU Aiqiong;XIE Zhiqin;YU Huaying;XIE Zhixun(College of Animal Science and Technology,Guangxi University,Nanning,Guangxi 530004;Guangxi Key Laboratory of Veterinary Biotechnology,Key Laboratory of China(Guangxi)-ASEAN Cross-border Animal Disease Prevention and Control of Ministry of Agriculture and Rural Affairs of China,Guangxi Veterinary Research Institute,Nanning,Guangxi 530001)

机构地区：[1]广西大学动物科学技术学院,广西南宁530004 [2]广西壮族自治区兽医研究所,农业农村部中国(广西)-东盟跨境动物疫病防控重点实验室,广西兽医生物技术重点实验室,广西南宁530001

出　　处：《中国家禽》2025年第3期85-92,共8页China Poultry

基　　金：广西科技基地和人才专项(桂科AD17195083);广西兽医生物技术重点实验室自主研究项目(24-035-32-A-01);广西八桂学者专项(2019A50)。

摘　　要：研究旨在建立一种快速检测圆圈病毒人源1型(GyH1)的方法。试验通过比对GenBank中GyH1基因组序列,基于序列保守区设计特异性引物和探针,经过筛选和优化,建立GyH1实时荧光重组酶介导核酸等温扩增(RAA)检测方法。结果显示:建立的实时荧光RAA方法可在20 min内鉴定GyH1;该方法特异性强,与圆圈病毒禽源1型(GyVg1)、鸡传染性贫血病毒(CIAV)、血清4型禽腺病毒(FAdV-4)、H9亚型禽流感病毒(H9 AIV)、新城疫病毒(NDV)、禽传染性支气管炎病毒(IBV)、传染性法氏囊病病毒(IBDV)、鸡细小病毒(ChPV)、J亚群禽白血病病毒(ALV-J)、鸡毒支原体(MG)、禽滑液囊支原体(MS)无交叉反应;敏感性高,筛出的引物探针可有效扩增,优化反应条件后检测限可达10~2拷贝/μL;重复性好,批内和批间变异系数均小于4%。采用该方法与荧光定量PCR方法同时对231份样品进行检测,两者检测结果相符。研究表明,成功建立了实时荧光RAA方法,可用于GyH1的鉴别检测,为基层快速检测GyH1提供了新的技术手段。The study was aimed at establishing a rapid detection method for gyrovirus homsa1(GyH1).The specific primers and probe were designed based on the conserved regions of GyH1 by searching and comparing sequences available in GenBank.After screening and optimization,the real-time fluorogenic recombinase acid amplication(RAA)detection method for GyH1 was established.The results showed that GyH1 could be identified in 20 minutes by the real-time fluorogenic RAA assay.This method had strong specificity and there was no cross-reaction with gyrovirus galga1(GyVg1),chicken infectious anemia virus(CIAV),fowl adenovirus serotype 4(FAdV-4),H9 subtype avian influenza virus(H9 AIV),Newcastle disease virus(NDV),avian infectious bronchitis virus(IBV),infectious bursal disease virus(IBDV),chicken parvovirus(ChPV),avian leukosis virus subgroup J(ALV-J),Mycoplasma gallisepticum(MG)and Mycoplasma synoviae(MS).The sensitivity was high,the screened primers and probe could be effectively amplified and the detection limit reached 102 copies/μL by optimizing the reaction conditions.The stability was good and the coefficient of variations in intra-and inter-assay were both less than 4%.Two hundred and thirty-one samples were tested simultaneously by real-time fluorogenic RAA and fluorescence quantitative PCR,and the results were consistent with each other.These results indicated that the real-time fluorogenic RAA method was successfully established for identification of GyH1,which provided a new technical means for the rapid field detection of GyH1.

关 键 词：圆圈病毒人源1型 重组酶介导核酸等温扩增 快速检测 即时检测 

分 类 号：S855.3[农业科学—临床兽医学]