禽呼肠孤病毒分子信标LAMP检测方法的建立  

Development of molecular beacon LAMP detection method for avian reovirus

作　　者：任红玉 谢芝勋[1] 华俊[1] 王盛[1] 万丽军 谢丽基[1] 谢志勤[1] 黄娇玲 张艳芳[1] 曾婷婷[1] 罗思思[1] HUREN Hongyu;XIE Zhixun;HUA Jun;WANG Sheng;WAN Lijun;XIE Liji;XIE Zhiqin;HUANG Jiaoling;ZHANG Yanfang;ZENG Tingting;LUO Sisi(Guangci Key Laboratory of Veterinary Biotechnology,Guangai Veterinary Research Institute,Nanning 530001,China)

机构地区：[1]广西兽医研究所广西兽医生物技术重点实验室,广西南宁530001

出　　处：《中国兽医学报》2023年第7期1402-1407,共6页Chinese Journal of Veterinary Science

基　　金：广西八桂学者专项资助项目(2019A50);国家万人计划科技创新领军人才专项资助项目(W02060083)。

摘　　要：为了建立一种快速鉴别诊断禽呼肠孤病毒(avian reovirus, ARV)的分子信标环介导等温扩增(LAMP)检测方法,根据ARV S1基因序列的高保守区域设计1套LAMP引物,并取F1c和B1c之间的序列设计分子信标(在5′端标记Cy5荧光基团,3′端标记BHQ-3淬灭基团),通过实时浊度仪,并对反应体系和反应条件进行优化,试验结果通过多通道荧光成像仪进行观察,结果显示:ARV阳性样品在荧光成像仪下显示红色荧光,而其他几种病原样品无荧光显示,建立的ARV分子信标LAMP检测方法仅特异性扩增ARV,与其他几种禽类常见疾病无交叉反应;对ARV的最低检测值为10拷贝/μL;在对22份临床样品的检测中,结果与荧光定量PCR的检测结果一致,将阳性产物进行测序鉴定均为ARV阳性样品。研究建立的ARV分子信标LAMP检测方法,结果可视化,整个试验流程无需开盖,能有效避免气溶胶污染,真正做到了特异性强、灵敏性好、准确性高,可应用于ARV的临床检测。To establish a molecular beacon LAMP detection method for rapid identification of avian reovirus(ARV),a set of LAMP primers according to the conserved sequence of the ARV S1 gene were designed.The molecular beacon was designed between F1c and B1c,and modifed with a Cy5 fluorophore group at the 5′end and a BHQ-3 quencher at the 3′end.The reaction systerm and condition were optimaled by turbidimeter.The reaction results were observed by multi-channel fluorescence imager,and showed that ARV-positive sample was red,while other poultry pathogen samples were not detected and showed no fluorescence,this detection method had no specific amplification for other avian pathogens.The minimum detection limit for ARV was 10 copies/μL.22 clinical samples were detected by the molecular beacon LAMP detection method,and the results were consistent with real-time quantitative PCR.The positive products were identified as ARV by sequencing.The results of the ARV molecular beacon LAMP detection method are visualized.And the whole process has a short cycle and does not need to open the lid,which can effectively avoid aerosol pollution.it truly has the advantages of good specificity and sensitivity and accuracy,and can be applied to the clinical detection of ARV.

关 键 词：禽呼肠孤病毒(ARV) 环介导等温扩增技术(LAMP) 分子信标 

分 类 号：S855.3[农业科学—临床兽医学]