猪瘟病毒和牛病毒性腹泻病毒双重荧光RT-PCR检测方法的建立
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天津海关科技计划项目(TK001-2017);国家质检公益性行业科研专项(201510017-02);国家质检总局科技计划项目(2015IK004)


Establishment of a Duplex Fluorescent RT-PCR Assay for Detection of Classical Swine Fever Virus and Bovine Viral Diarrhea Virus
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    摘要:

    为同时检测猪源临床样本中的猪瘟病毒(CSFV)和牛病毒性腹泻病毒(BVDV),基于这两种病毒的5'UTR序列设计特异引物和TaqMan探针,建立了一种检测CSFV和BVDV的双重荧光RT-PCR检测方法,并对该方法的特异性、最低检出限和重复性等进行了评价。结果显示,该方法只对CSFV和BVDV呈现特异性扩增,对猪伪狂犬病病毒、猪繁殖与呼吸综合征病毒、猪传染性胃肠炎病毒、猪流行性腹泻病毒、猪圆环病毒2型不发生交叉反应,对阳性标准对照CSFV-5'UTR-RNA、BVDV-1-5'UTR-RNA和BVDV-2-5'UTR-RNA,最低可分别检出27、36和32拷贝/μL。该方法的组内和组间试验Ct值变异系数介于0.11%~1.20%,具有良好的重现性。对152份猪组织样本用该方法进行CSFV和BVDV核酸检测,结果检出CSFV阳性样本16份,BVDV阳性样本3份,CSFV和BVDV双阳性样本1份,与国标和OIE《陆生动物诊断试验和疫苗》相应的荧光RT-PCR方法阳性符合率为100%。结果表明,本研究建立的双重荧光RT-PCR方法可用于临床样本中的CSFV和BVDV检测,从而为猪瘟防制和净化提供了一种有效的技术手段。

    Abstract:

    In order to simultaneously detect classical swine fever virus(CSFV)and bovine viral diarrhea virus(BVDV)in clinical samples from pigs,the specific primers and TaqMan probes were designed based on 5'UTR sequence of the two kinds of viruses,and a duplex fluorescent RT-PCR was established,the specificity,minimum detection limit and repeatability were evaluated. The results showed that the assay could react specifically with CSFV and BVDV only,but failed to crossly react with other viruses including pseudorabies virus(PRV),porcine reproductive and respiratory syndrome virus(PRRSV),transmissible gastroenteritis virus(TGEV),porcine epidemic diarrhea virus(PEDV)and porcine circovirus-2(PCV-2). The minimum detection limits were 27,36 and 32 copies/μL for the positive standard plasmid control of CSFV-5'UTR-RNA,BVDV-1-5'UTR-RNA and BVDV-2-5'UTR-RNA respectively. The coefficients of variation(CV)of intra-and inter-group ranged 0.11% to 1.20%,showing good reproducibility. Atotal of 152 tissue samples were tested for CSFV and BVDV by the assay,with the results of 16 CSFV positive samples,3 BVDV positive samples and 1 CSFV-BVDV dual positive samples,which were completely consistent with the national standard and the results of corresponding fluorescent RT-PCR assay specified in OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. In conclusion,the assay established in this study could be used for the detection of CSFV and BVDV in clinical samples,which provided an effective technical method for control and purification of the two diseases.

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张宏伟,王建华,陈本龙,麻丽丹,宋喆,蒲静.猪瘟病毒和牛病毒性腹泻病毒双重荧光RT-PCR检测方法的建立[J].《中国动物检疫》编辑部,2020,37(9):106-112.

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  • 在线发布日期: 2020-09-02
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