新型阿卡斑病毒SYBR Green Ⅰ荧光定量RT-PCR建立及广西边境地区蚊携带新型阿卡斑病毒调查  

Development of qRT-PCR based on novel Akabane virus SYBR Green Ⅰ and survey on novel Akabane virus carried by mosquitoes in border areas of Guangxi

作　　者：马玲[1] 孟菲[1] 陆晨阳 陈赫威 蔡畅 秦树英 覃绍敏[1] 刘金凤[1] 陈凤莲[1] 吴健敏[1] MA Ling;MENG Fei;LU Chen-yang;CHEN He-wei;CAI Chang;QIN Shu-ying;QIN Shao-min;LIU Jin-feng;CHEN Feng-lian;WU Jian-min(Guangxi Veterinary Research Institute/Guangxi Key Laboratory of Veterinary Biotechnology,Nanning,Guangxi 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：《南方农业学报》2022年第6期1734-1741,共8页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31260830);广西兽医生物技术重点实验室开放基金项目(20-065-23-B-2);广西兽医研究所基本科研业务费专项(桂科专项21-7)。

摘　　要：【目的】建立检测新型阿卡斑病毒(AKAV)的SYBR Green Ⅰ荧光定量RT-PCR,并结合序列测定开展广西边境地区蚊携带新型AKAV调查,了解其流行趋势及遗传进化特征,为建立重要蚊媒病毒病预警机制提供技术支持。【方法】分别针对新型AKAV的S基因和M基因主要变异位点设计引物,建立SYBR Green Ⅰ荧光定量RT-PCR并通过Ct值、标准曲线斜率、相关系数、扩增效率及扩增准确性等指标确定最佳反应体系及扩增程序;以优化的SYBR Green Ⅰ荧光定量RT-PCR检测2019年6-10月在广西边境地区采集的139份蚊样品,获得的新型AKAV经测序后采用ClustalW构建遗传进化树。【结果】SYBR Green Ⅰ荧光定量RT-PCR最佳反应体系:SYBR Premix Ex Taq Ⅱ 10.0μL,上、下游引物(4μmol/L)各1.0μL,标准品或反转录产物2.0μL,双蒸水补齐至20.0μL。扩增程序:95℃预变性30 s;95℃30 s,64℃(S基因)/66℃(M基因)30 s,72℃30 s,进行40个循环。SYBR Green Ⅰ荧光定量RT-PCR检测新型AKAV S基因和M基因的极限为1.0×101copies/μL和1.0×102copies/μL,对应的扩增效率分别为98.61%和105.81%,但不能扩增蓝舌病病毒、鹿流行性出血热病毒、竹鼠圆环病毒和布鲁氏杆菌;检测S基因和M基因的批内重复试验、批间重复试验变异系数(CV)均低于5.00%。采用建立的SYBR Green I荧光定量RT-PCR成功从广西边境地区防城港市、东兴市、宁明县、大新县及北海市的阿蚊和库蚊中检出新型AKAV,且均属于基因Ia型,尤其与广东和湖南蠓虫、中华按蚊、致倦库蚊分离毒株具有较高的相似性。【结论】针对新型AKAV S基因和M基因建立的SYBR Green Ⅰ荧光定量RT-PCR具有特异性强、灵敏度高、重复性好的特点,更适用于检测病毒拷贝数低的样品。此外,从广西边境地区蚊样品中检出的AKAV毒株均为新型AKAV,属于基因Ia型,与广东和湖南蠓虫、中华按蚊、致倦库蚊分离毒株具有较高的相似性,推测新型AKAV【Objective】To develop real-time reverse transcription-quantitative polymerase chain reaction(qRT-PCR)for the identification of novel Akabane vrius(AKAV)SYBR Green Ⅰ,to find out the prevalence and variation of in mosquito-carried novel AKAV in the border areas of Guangxi through sequencing,so as to provide technical support for warning mechanism of mosquito-borne viruses.【Method】Primers were designed based on main variation sites of S gene and M gene in AKAV for establish q RT-PCR of SYBR Green Ⅰ.According to Ct,slope of standard curve,correlation coefficient,amplification efficiency and precision,optimal response system and amplification procedure,and the optimal qRT-PCR for SYBR Green Ⅰ was used to test 139 mosquito samples collected from 8 sampling sites in border areas of Guangxi during June to October of 2019.After acquiring novel AKAV,its phylogenetic tree was constructed using ClustalW.【Result】The q RT-PCR methods for SYBR Green Ⅰ were optimized in 10.0μL of SYBR Premix Ex Taq Ⅱ,1.0μL of forward primers(4μmol/L),1.0μL of reverse primers(4μmol/L),2.0μL of template or reverse transcription products,and making up to 20.0μL volume with ddHO.Amplification procedure:95℃initial denaturation 30 s,95℃30 s,64℃(S gene)/66℃(M gene)30 s,72℃30 s for 40 cycles.The detection limits of SYBR Green Ⅰ qRT-PCR methods for detecting S gene and M gene in AKAV were 1.0×10~1copies/μL and 1.0×10~2copies/μL,respectively;and the amplification efficiency for detecting S gene and M gene in AKAV were 98.61%and 105.81%,respectively.But such method was unable to amplify bluetongue virus,epidemic hemorrhagic fever of deer virus,bamboo rat circovirus and Brucella,and coefficient of variation(CV)of and intra-batch and inter-batch repeated test were all below 5.00%.With the newly-established qRT-PCR for SYBR Green Ⅰ,novel AKAV was detected in Armigeres subalbatus and culices from Fangchenggang City,Dongxing City,Ningming County,Daxin County and Beihai City in border areas of Guangxi.Novel AKAV,bel

关 键 词：新型阿卡斑病毒(AKAV) 蚊媒病毒 SYBR GreenⅠ荧光定量RT-PCR 广西边境地区 

分 类 号：S852.659.5[农业科学—基础兽医学]