蓝舌病病毒群特异性RT-LAMP检测方法的建立与初步应用  

Establishment and Application of a Group Specific Reverse Transcriptase Loop-mediated Isothermal Amplification Method of Bluetongue Virus

作　　者：李占鸿 朱沛 宋子昂 李卓然 杨振兴 李华春 杨恒 廖德芳 LI Zhanhong;ZHU Pei;SONG Ziang;LI Zhuoran;YANG Zhenxing;LI Huachun;YANG Heng;LIAO Defang(Yunnan Tropical and Subtropical Animal Virus Diseases Laboratory, Yunnan Animal Science and Veterinary Institute, Kunming 650224, China;College of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, China)

机构地区：[1]云南省畜牧兽医科学院,云南省热带亚热带动物病毒重点实验室,昆明650224 [2]云南农业大学动物医学院,昆明650201

出　　处：《畜牧兽医学报》2021年第8期2244-2253,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：云南省科技厅科技计划项目-青年项目(2018FD002);国家重点研发计划(2017YFC1200505,2016YFD0500908);公益性行业(农业)科研专项(201303035)。

摘　　要：本研究旨在建立针对中国流行蓝舌病病毒(bluetongue virus,BTV)的逆转录-环介导等温扩增(RT-LAMP)检测方法。根据我国分离BTV毒株Seg-5基因序列的高度保守区域,设计特异性引物,优化反应条件及反应体系,进行特异性及灵敏度验证,建立BTV RT-LAMP检测方法;对46株中国分离的12种血清型BTV(BTV-1、-2、-3、-4、-5、-7、-9、-12、-15、-16、-21与-24)代表毒株进行检测,进一步对120份BTV核酸阳性血液样品及60份2020年采集自监控动物的临床血液样品进行BTV的RT-LAMP及qRT-PCR检测,验证建立的RT-LAMP检测方法的准确性和可靠性。试验结果表明,本研究建立的RT-LAMP方法最佳反应条件为64℃,扩增45 min;反应体系中外引物∶内引物:环引物的最佳浓度及比例为0.2μmol·L^(-1)∶0.6μmol·L^(-1)∶0.4μmol·L^(-1)。该方法可特异性检测中国流行的12种血清型BTV核酸,与动物流行性出血病病毒(EHDV)、中山病毒(CHUV)、阿卡斑病毒(AKAV)、口蹄疫病毒(FMDV)及非洲马瘟病毒(AHSV)核酸均无交叉扩增现象;最低可检测4.5拷贝·μL^(-1)的BTV核酸。对46株分属12种血清型的BTV代表毒株的检测结果均为阳性;120份BTV核酸阳性血液样品的检测结果与qRT-PCR检测结果无显著差别(McNemar检验P>0.05),符合率为95.0%;60份临床血液样品的检测结果与qRT-PCR结果完全一致,以上结果表明本研究建立的RT-LAMP方法准确可靠,对中国分离的BTV毒株及临床血液样品均具有良好的检测效果。本研究建立的BTV RT-LAMP检测方法具有反应快速、结果可视化、特异性强和敏感度高等优点,为我国开展BTV检测诊断与流行病学研究提供了技术支持,具有较好的应用前景。The purpose of this study was to establish a reverse transcription loop-mediated isothermal amplification(RT-LAMP)method for the detection of bluetongue viruses(BTVs)prevalent in China.Specific primers were designed according to the highly conserved regions of the Seg-5 gene of BTV strains isolated in China.By optimizing the reaction condition,the RT-LAMP method was established,and the specificity and sensitivity of the method were evaluated.To verify the accuracy and reliability of the RT-LAMP method,46 BTV strains belonging to 12 sero-types(BTV-1,-2,-3,-4,-5,-7,-9,-12,-15,-16,-21 and-24)were tested,further 120 BTV nucleic acid-positive blood samples collected from BTV-infected animals and 60 blood samples collected from sentinel animals in 2020 were tested through RT-LAMP and qRT-PCR simultaneously.The optimal reaction temperature of the RT-LAMP was 64℃,and the optimal reaction time was 45 min,the optimal concentration and ratio of the outer primer:inner primer:loop primer in the reaction mixture was 0.2μmol·L^(-1):0.6μmol·L^(-1):0.4μmol·L^(-1).This method can specifically detect the nucleic acids of BTV belonged to the 12 epidemic serotypes in China without any cross-reaction with the nucleic acids of epidemic hemorrhagic virus(EHDV),Chuzan disease virus(CHUV),Akabane disease virus(AKAV),foot-and-mouth disease virus(FMDV)and African horse sickness virus(AHSV).The lower detecting limit of the method was 4.5 copi-es·μL^(-1) of BTV genomes.The detection results of 46 BTV strains belonged to 12 serotypes were positive.There was no significant difference(McNemar test P>0.05)between the detection results of RT-LAMP and qRT-PCR for 120 BTV nucleic acid positive blood samples with a coincidence rate of 95.0%.The detection results of 60 blood samples were completely consistent between RT-LAMP and qRT-PCR.These data demonstrated that the RT-LAMP method established in this study was accurate and reliable to detect the BTV strains isolated in China and the clinical blood samples collected from animals.The BTV RT

关 键 词：蓝舌病病毒 群特异性 RT-LAMP 应用 

分 类 号：S852.659.4[农业科学—基础兽医学]