為建立一種快速檢測1型、2型牛病毒性腹瀉病毒（BVDV）的通用RT-PCR方法，根據(jù)GenBank上收錄的64株1型、2型BVDV以及1株豬瘟病毒（CSFV）的全基因組序列，應用Primer 6.0軟件設計針對5'-UTR區(qū)域的1型、2型BVDV特異性通用引物對，擴增目的片段，并對該方法進行特異性、敏感性、重復性試驗及利用該方法開展臨床樣品檢測。結果顯示：擴增的目的片段長度約為302 bp；該方法的靈敏度為2.09×102 copies/μL，無非特異性擴增，且重復性良好。對采自山東省發(fā)病牛場的13份鼻腔棉拭子和9份牛血清臨床樣品進行檢測，發(fā)現(xiàn)有8份鼻腔棉拭子和8份血清為BVDV陽性，隨機抽取4份陽性樣品送測序，發(fā)現(xiàn)3份樣品毒株為1型BVDV，1份樣品還需進一步鑒定。本研究建立的RT-PCR方法可實現(xiàn)對1型、2型BVDV核酸的特異性檢測，也可用于該病的流行病學調(diào)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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