中国蓝舌病病毒流行株血清型实时荧光定量RT-PCR检测方法的建立与应用

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

【背景】蓝舌病病毒(Bluetongue virus,BTV)是一种严重危害反刍动物的虫媒病毒,我国存在12种血清型BTV(BTV-1、-2、-3、-4、-5、-7、-9、-12、-15、-16、-21和-24)的流行。【目的】建立12种血清型BTV的RT-qPCR定型方法,为BTV的诊断与流行病学研究提供技术保障。【方法】根据我国流行BTV基因节段2(Seg-2)序列设计引物和TaqMan探针,对引物的特异性与敏感性进行评估;以12种血清型BTV毒株和核酸阳性血液样本验证建立的血清型RT-qPCR检测方法;将其应用于库蠓与动物血液样本中BTV的定型。【结果】建立的BTV血清型RT-qPCR检测方法具有良好的特异性与灵敏性,反应的扩增效率(E)值>90.3%,相关系数(R2)值在0.991-0.999之间,对12种血清型BTV核酸的检测下限在25-48拷贝之间。对165株BTV的RT-qPCR定型结果与病毒的Seg-2测序鉴定结果一致;对194份采集于哨兵动物的BTV核酸阳性血液样本的RT-qPCR定型结果与感染动物上分离BTV的血清型一致。采用建立的方法,从2019年云南省师宗县与景洪市采集的库蠓与牛血液样本中鉴定出6种血清型的BTV(BTV-1、-2、-4、-5、-16和-24)。【结论】研究建立的12种BTV血清型RT-qPCR定型方法具有特异、敏感和省时的优点,可用于媒介与动物感染BTV的血清型定型,具有良好的应用与推广价值。

[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.