阿卡斑病毒荧光RT-RAA快速检测方法的建立及初步应用  

Establishment and preliminary application of fluorescent RT-RAA for rapid detection of Akabane virus

作　　者：宋瑞鹏 刘金凤 钟华 孙倩 陈赫威 陈凤莲 覃绍敏 林俊 陆晨阳 秦树英 许力士 韦珊珊 马玲 韦珏 吴健敏[1,2] SONG Rui-peng;LIU Jin-feng;ZHONG Hua;SUN Qian;CHEN He-wei;CHEN Feng-lian;QIN Shao-min;LIN Jun;LU Chen-yang;QIN Shu-ying;XU Li-shi;WEI Shan-shan;MA Ling;WEI Jue;WU Jian-min(College of Animal Science and Technology,Guangxi University,Nanning 530004,China;Guangxi Key Laboratory of Veterinary Biotechnology,Key Laboratory of China(Guangxi)-ASEAN Cross-border Animal Disease Prevention and Control,Ministry of Agriculture and Rural Affairs of China,Guangxi Veterinary Research Institute,Nanning 530001,China;College of Marine Sciences and Biotechnology,Guangxi University for Nationalities,Nanning 530006,China)

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广西兽医生物技术重点实验室,农业农村部(广西)东盟跨境动物疫病防控重点实验室,广西南宁530001 [3]广西民族大学海洋与生物技术学院,广西南宁530006

出　　处：《中国兽医科学》2023年第5期552-558,共7页Chinese Veterinary Science

基　　金：国家自然科学基金项目(32160830);广西兽医生物技术重点实验室项目(19-50-40-B-03);广西基本科研业务费(桂科专项21-7)。

摘　　要：为建立一种快速检测阿卡斑病毒(AKAV)的分子生物学检测方法,本研究通过比较分析AKAV基因组序列,在S基因保守序列区域设计重组酶介导等温扩增(RAA)引物及探针,筛选最佳引物对,然后对RAA反应温度、引物浓度以及探针浓度进行优化,建立了AKAV的荧光逆转录重组酶介导的核酸等温扩增(RT-RAA)快速检测方法。结果显示,所建立的荧光RT-RAA方法在42℃反应20 min即可快速检测出AKAV,检测下限可达6.26×10^(1)copies/μL,与牛病毒性腹泻病毒、蓝舌病毒、伪狂犬病病毒、猪繁殖与呼吸障碍综合征病毒等无交叉反应,并具有良好的重复性。对37份临床样品进行检测,阳性检出率为43.24%,与RT-qPCR检测结果符合率为94.59%。结果表明,建立的AKAV荧光RT-RAA快速检测方法具有快速、特异、灵敏、稳定等特点,可为AKAV的现场快速检测提供可靠方法。The purpose of this study is to establish a rapid molecular biological detection method for detecting Akabane virus(AKAV),recombinase-mediated isothermal amplification(RAA) primers and probes were designed in the conserved sequence region of the S gene by comparative analysis of AKAV genome sequences.In order to screen the best primer pairs,after which the RAA reaction temperature,primer concentration and probe concentration were optimized,and a rapid fluorescent reverse transcriptase-mediated isothermal amplification of nucleic acids(RT-RAA)assay for AKAV was established.The results show that the established RT-RAA method can rapidly detect AKAV at 42℃ in 20 min,with a detection limit lower to 6.26×10^(1) copies/μL.It is non-cross-reactive with bovine viral Diarrhea virus,bluetongue virus,pseudorabies virus,porcine reproductive and respiratory disorder syndrome virus bovine and swine viruses,and has good reproducibility.The positive rate of 37 clinical samples tested using this method for detection was 43.24%,with the results of the coincidence rate with RT-q PCR was 94.59%.In conclusion,the AKAV fluorescence RT-RAA assay is rapid,specific,sensitive and stable,and can be a reliable method for the rapid detection of AKAV in the field.

关 键 词：阿卡斑病毒 重组酶介导等温扩增 快速检测 

分 类 号：S852.659.5[农业科学—基础兽医学]