Development and application of a real-time RT-PCR for serotyping of bluetongue virus strains epidemic in China

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.

Key words:Bluetongue virus;serotype;RT-qPCR;application