BHV-1、BRSV、BPIV-3和BVDV四重荧光PCR检测方法的建立与应用
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S854.4

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湖南省草食动物体系岗位专家基金项目


Establishment and Application of Quadruple Fluorescence PCR Assay for Detection of BHV-1,BRSV,BPIV-3 and BVDV
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    摘要:

    为建立检测牛疱疹病毒(BHV-1)、牛呼吸道合胞体病毒(BRSV)、牛副流感病毒3型(BPIV-3)和牛病毒性腹泻病毒(BVDV)单一或混合感染的荧光PCR检测方法,根据BHV-1 gB基因、BRSV F基因、BPIV-3 M基因和BVDV 5'UTR基因保守区序列分别设计特异性引物和TaqMan荧光探针,经条件优化,成功建立了BHV-1、BRSV、BPIV-3和BVDV的四重荧光PCR检测方法。该方法对牛布鲁氏菌、猪瘟病毒、小反刍兽疫病毒、羊多杀性巴氏杆菌无特异性扩增;对BHV-1、BPIV-3和BVDV的最低检测量均为8.268 copies/μL,对BRSV的最低检测量为82.680 copies/μL;该方法重复性好,CV值为1%~2%。应用本方法检测采自湖南省内某屠宰场的865份样品,结果BHV-1、BRSV、BPIV-3和BVDV等4种病原均有检出,其阳性率分别为0.58%,0.81%,0.23%和0.81%。本研究建立的多重荧光PCR检测方法可同时对BHV-1、BRSV、BPIV-3和BVDV进行检测,为这4种病原的快速诊断和鉴别提供了技术支撑。

    Abstract:

    In order to establish a fluorescent PCR assay for detection of single or mixed infection of bovine herpesvirus type 1(BHV-1),bovine respiratory syncytial virus(BRSV),bovine parainfluenza virus type 3(BPIV-3)and bovine viral diarrhea virus(BVDV),specific primers and TaqMan fluorescent probes were respectively designed according to the conserved region sequences of BHV-1 gB gene,BRSV F gene,BPIV-3 M gene and BVDV 5'UTR,followed by optimization of conditions,a quadruple fluorescence PCR assay was successfully established for BHV-1,BRSV,BPIV-3 and BVDV. The results showed that no specific amplification by the method for Brucella bovis,classical swine fever virus(CSFV),peste des petits ruminants virus(PPRV)and Pasteurella multocida was detected,and the minimum detectable amounts were all 8.268 copies/μL for BHV-1,BPIV-3 and BVDV,and 82.680 copies/μL for BRSV;the CV value of the method was 1% to 2% due to its good repeatability. By the method,BHV-1,BRSV,BPIV-3 and BVDV were all detected out from 865 samples collected from a slaughterhouse in Hunan Province,with the positive rates of 0.58%,0.81%,0.23% and 0.81% respectively. In conclusion,the method established in the paper could simultaneously detect BHV-1,BRSV,BPIV-3 and BVDV,which technically supported rapid diagnosis and identification for the above pathogens.

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姜利霞,王玉海,何世成,胡巧云,王卫国,鲁杏华,武维保,谢怡灵,王昌建. BHV-1、BRSV、BPIV-3和BVDV四重荧光PCR检测方法的建立与应用[J].《中国动物检疫》编辑部,2021,38(10):98-106.

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