Abstract:[Background] Bluetongue virus (BTV), one kind of arbovirus, causes seriously harms in ruminants and twelve serotypes of BTV (BTV-1, -2, -3, -4, -5, -7, -9, -12, -15, -16, -21 and -24) are widely prevalent in China. [Objective] To provide technical support for the diagnosis and epidemiology research of BTV, we aimed to develop serotyping RT-qPCR method for 12 BTV serotypes which were epidemic in China. [Methods] Primers and TaqMan probes based on the Seg-2 sequences of Chinese BTVs were designed and their specificity and sensitivity were evaluated. The reliability of the established RT-qPCR method was assessed with BTV strains and BTV-positive blood samples then further applied to identify the serotypes of BTV in Culicoides and blood samples. [Results] The established BTV serotype RT-qPCR method showed highly specificity and sensitivity with the amplification efficiency (E) values larger than 90.3%, the correlation coefficient values (R2) ranging from 0.991 to 0.999 and the minimum copy number of detectable BTV nucleic acid ranging from 25 to 48 copies. Serotype identification of 165 isolated BTV strains by RT-qPCR and Seg-2 sequencing showed consistent results. Furthermore, serotype RT-qPCR identification results of 194 BTV-positive blood samples from infected sentinel animals were consistent with serotyping results of the isolated BTVs. Using the established RT-qPCR method, six serotypes of BTV (BTV-1, -2, -4, -5, -16 and -24) were identified from the Culicoides and cattle blood samples collected from Shizong county and Jinghong district of Yunnan province. [Conclusion] The BTV serotype RT-qPCR established here could be used for diagnosis the serotypes of BTV in vectors and animals with advantage of time saving, high specificity and sensitivity.
