科研动态

为建立一种快速检测1型、2型牛病毒性腹泻病毒（BVDV）的通用RT-PCR方法，根据GenBank上收录的64株1型、2型BVDV以及1株猪瘟病毒（CSFV）的全基因组序列，应用Primer 6.0软件设计针对5'-UTR区域的1型、2型BVDV特异性通用引物对，扩增目的片段，并对该方法进行特异性、敏感性、重复性试验及利用该方法开展临床样品检测。结果显示：扩增的目的片段长度约为302 bp；该方法的灵敏度为2.09×102 copies/μL，无非特异性扩增，且重复性良好。对采自山东省发病牛场的13份鼻腔棉拭子和9份牛血清临床样品进行检测，发现有8份鼻腔棉拭子和8份血清为BVDV阳性，随机抽取4份阳性样品送测序，发现3份样品毒株为1型BVDV，1份样品还需进一步鉴定。本研究建立的RT-PCR方法可实现对1型、2型BVDV核酸的特异性检测，也可用于该病的流行病学调查研究。

Establishment and Application of a General RT-PCR Assay for Detection of BVDV Type 1 and 2

In order to establish a general RT-PCR assay to rapidly detect bovine viral virus（BVDV）type 1 and 2，the general primer was designed based on 5'UTR sequences of BVDV type 1 and 2 by Primer 6.0 software according to the analysis of complete gene sequence of 64 strains of BVDV type 1and 2 and one strain of classical swine fever virus（CSFV）registered in GenBank，and the target fragment was amplified，then the specificity，sensitivity and reproducibility of the RT-PCR method were evaluated，and the clinical samples were tested. The results showed that the amplified target fragment was about 302 bp；the detection limt of the method was 2.09×102 copies/μL，with no nonspecific amplification but good reproducibility. 13 nasal swabs and 9 bovine serum samples collected from an infected farm in Shandong Province were detected by the established method，it was found that 8 nasal swabs and 8 sera were positive against BVDV，then 4 of the positive samples were randomly selected to carry out virus gene sequencing and comparison，it was found that virus of 3 samples could be identified as BVDV type 1，and the other 1 sample still needed further analysis. In conclusion，specific detection of nucleic acids of BVDV 1 and 2 could be realized by the RT-PCR assay established in the study，which could also be used in epidemiological investigation of BVDV.

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