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

【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 sinensis，Culex quinquefasciatu，which can be inferred that novel AKAV has already spread in Guangxi and surrounding provinces.