牛环曲病毒RT-PCR检测方法的建立及应用

Establishment and application of RT-PCR method for detection of bovine torovirus

为建立牛环曲病毒快速检测方法,针对牛环曲病毒的保守区域S基因设计合成1对特异性引物,建立了检测牛环曲病毒的RT-PCR方法,并对其进行了敏感性、特异性和重复性检测。结果显示,仅牛环曲病毒阳性模板扩增得到698bp大小的目的条带,测序结果与GenBank中牛环曲病毒S基因序列(登录号:MG957145.1)的同源性为97%。敏感性试验显示,该方法能检测到的最低核酸含量为1.36×10^4 copies/μL。利用该方法对采集自河南省部分地区的164份临床样品进行检测,结果显示牛环曲病毒的检出率为9.15%(15/164)。结果表明,所建立的牛环曲病毒RTPCR检测方法灵敏度高、特异性强、重复性好,可用于牛环曲病毒的临床检查和流行病学调查。

To establish a rapid RT-PCR method for detection of bovine torovirus(BToV),apair of special primers was designed based on the conserved regions of BToV S gene.And its sensitivity,specificity and repeatability were tested.The results indicated that only BToV was successfully amplified a target fragment of 698bp in size.When compared the sequence from the PCR products with other BToV sequences in GenBank,they showed 97%identity.Testing of the sensitivity of RT-PCR indicated that the lowest detection limit was 1.36×10^4 copies/μL nuclear acids.164fecal samples collected from different farms in Henan Province were detected by the established method,and the positive rate was 9.15%.The results showed that we successfully established the RTPCR assay with high sensitivity,strong specificity and good repeatability to detect bovine torovirus,which can apply the clinic detection and molecular epidemiological survey.