1型、2型牛病毒性腹泻病毒通用RT-PCR检测方法的建立与应用
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Establishment and Application of a General RT-PCR Assay for Detection of BVDV Type 1 and 2
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    摘要:

    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。

    Abstract:

    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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曾维欢,李蓉,肖望成,康京丽,黄保续,吴发兴,代飞燕.1型、2型牛病毒性腹泻病毒通用RT-PCR检测方法的建立与应用[J].《中国动物检疫》编辑部,2021,38(2):98-103.

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  • 在线发布日期: 2021-02-01
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