為建立一種快速檢測(cè)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(yīng)用Primer 6.0軟件設(shè)計(jì)針對(duì)5'-UTR區(qū)域的1型、2型BVDV特異性通用引物對(duì),，擴(kuò)增目的片段,，并對(duì)該方法進(jìn)行特異性、敏感性,、重復(fù)性試驗(yàn)及利用該方法開展臨床樣品檢測(cè),。結(jié)果顯示：擴(kuò)增的目的片段長(zhǎng)度約為302 bp；該方法的靈敏度為2.09×102 copies/μL,，無非特異性擴(kuò)增,，且重復(fù)性良好。對(duì)采自山東省發(fā)病牛場(chǎng)的13份鼻腔棉拭子和9份牛血清臨床樣品進(jìn)行檢測(cè),，發(fā)現(xiàn)有8份鼻腔棉拭子和8份血清為BVDV陽(yáng)性,，隨機(jī)抽取4份陽(yáng)性樣品送測(cè)序，發(fā)現(xiàn)3份樣品毒株為1型BVDV,，1份樣品還需進(jìn)一步鑒定,。本研究建立的RT-PCR方法可實(shí)現(xiàn)對(duì)1型、2型BVDV核酸的特異性檢測(cè),，也可用于該病的流行病學(xué)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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