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

  • 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 10.0 μL,上、下游引物(4 μmol/L)各1.0 μL,标准品或反转录产物2.0 μL,双蒸水补齐至20.0 μL。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AKAVS基因和M基因的极限为1.0×101 copies/µL和1.0×102 copies/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已在广西周边省份流行。

     

    Abstract: 【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 qRT-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 qRTPCR 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 qRT-PCR methods for SYBR Green I were optimized in 10.0 μL of SYBR Premix Ex Taq II,1.0 μL of forward primers(4 μmol/L),1.0 μL of reverse primers(4 μmol/L),2.0 μL of template or reverse transcription products,and making up to 20.0 μL volume with ddH2O.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 1.0×101 copies/µL and 1.0×102 copies/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,belonging to genogroup Ia,were highly similar with the strains isolated from midges of Guangdong and Hunan Province,Anopheles sinensis,Culex quinquefasciatus.【Conclusion】 The SYBR Green I qRT-PCR based on S gene and M gene in novel AKAV had the advantage of specificity,precision,repeatability and high sensitivity,which works better for lower copy numbers of sample detection.Moreover,AKAV strains detected from mosquito samples from border areas in Guangxi were novel AKAV,and their strains belonged to genogroup Ia.It is highly similar with the strains isolated from midges of Guangdong and Hunan Province,Anopheles sinensisCulex quinquefasciatu,which can be inferred that novel AKAV has already spread in Guangxi and surrounding provinces.

     

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