Establishment of One Step qRT-PCR Detection Assays for Bluetongue virus Serotype 25, 26 and 27
LI Zhuo-Ran1,*, SONG Zi-Ang2,*, LI Zhan-Hong1, YANG Zhen-Xing1, YANG Heng1,**, LIAO De-Fang1,**
1 Yunnan Tropical and Subtropical Animal Virology Laboratory, Yunnan Academy of Animal Husbandry and Veterinary, Kunming 650224, China; 2 College of Veterinary Medicine, Yunnan Agricultural University, Kunming 650224, China
AbstractBluetongue virus (BTV) is an arbovirus that seriously endangers ruminants. Since 2008, 3 new serotypes of BTV-25, -26 and -27 have been discovered in the worldwide, and China is also threatened by the invasion of BTV-25, -26 and -27. The DNA sequences of BTV-25, -26 and -27 genomic segment 2 (Seg-2) with lengths of about 500 bp were synthesized and transcribed into single strand RNA (ssRNA) in vitro using as the reference nucleic acids. Serotype specific amplification primers and TaqMan probes were designed based on Seg-2 sequences of BTV-25, -26 and -27 and the one step qRT-PCR detection assays were established. The results showed that the reference nucleic acids of BTV-25, -26 and -27 were successfully obtained; The established BTV-25, -26 and -27 serotype specific one step qRT-PCR detection assays possessed strong specificity, high sensitivity and good repeatability, which did not cross-react with other BTV serotype virus, Epizootic haemorrhagic disease virus, Akabane virus and African horse sickness virus inactivated vaccine virus;and that showed the minimum ssRNA template detection limits of 4.43×101, 5.61×101 and 4.88×101 copies/μL for BTV-25, -26 and BTV-27 ssRNA, respectively; and displayed the intra- and inter-group coefficient of variation varying between 0.48% and 1.79%. To determine the presence of BTV-25, -26 and -27 infection, 120 BTV nucleic acid positive animal blood samples collected from southern China were tested, and the results were all negative, representing none of the animals were infected by BTV-25, -26 and -27. This study successfully established the one step qRT-PCR detection assays of BTV-25, -26 and -27, and provides an effective technical means for the monitoring of new serotype BTV intrusion into China.
LI Zhuo-Ran,SONG Zi-Ang,LI Zhan-Hong, et al. Establishment of One Step qRT-PCR Detection Assays for Bluetongue virus Serotype 25, 26 and 27[J]. 农业生物技术学报, 2020, 28(6): 1132-1140.
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