牛呼吸道合胞体病毒RT-LAMP检测方法的建立
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摘要
牛呼吸道合胞体病毒(BRSV)是引起牛呼吸道疾病的一种病毒性病原体。本研究建立一种利用环介导等温扩增技术(RT-LAMP)快速检测BRSV的新的方法,针对BRSV N基因(GeneBank:AF054668.1)设计4对特异性LAMP引物,每组引物特异性识别靶基因序列上4个独立区域,利用扩增靶DNA的Bst2.0 DNA聚合酶,63℃恒温水浴50 min完成病毒检测,使用钙黄绿素检测荧光目视和琼脂糖凝胶电泳方法,同时对牛的其他主要病毒进行特异性检测,结果证明特异性好。该RT-LAMP检测方法的灵敏度达到BRSV模板稀释的10~(-6)倍,可成功检测到临床样本中的BRSV基因组,为BRSV的临床检测提供了一种快速的试验手段,更适合BRS的诊断和临床现场快速检测。
BRSV is a severe viral infectious disease of bovine.A new method for rapidly detecting BRSV taking advantage of loop-mediated isothermal amplificatio was developed.The whole reaction lasted only 50 minutes at a constructed temperature of 63℃ using Bst2.0 DNA polymerase to amplificate target gene.The result was detected by calcein-AM fluorescence inspection and agarose gel electrophonesis,comparing with other major viral diseases of bovine,which confirmed its good specificity.The result demonstrated that the BRSV RT-LAMP assay can detect the BRSV when the template is diluted at least 10~(-6) times,which can successfully detect the BRSV of clinical samples.The rapid sensitive and special loop-mediated isothermal amplification assay may be a potential tool for the detection of BRSV in clinical and field testing.
BRSV is a severe viral infectious disease of bovine.A new method for rapidly detecting BRSV taking advantage of loop-mediated isothermal amplificatio was developed.The whole reaction lasted only 50 minutes at a constructed temperature of 63℃ using Bst2.0 DNA polymerase to amplificate target gene.The result was detected by calcein-AM fluorescence inspection and agarose gel electrophonesis,comparing with other major viral diseases of bovine,which confirmed its good specificity.The result demonstrated that the BRSV RT-LAMP assay can detect the BRSV when the template is diluted at least 10~(-6) times,which can successfully detect the BRSV of clinical samples.The rapid sensitive and special loop-mediated isothermal amplification assay may be a potential tool for the detection of BRSV in clinical and field testing.
引文
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[14] 李丽阳,刘洋,王磊,等.脂筏在牛呼吸道合胞体病毒感染MDBK细胞中的作用[J].中国兽医学报,2017,37(4):643-647.
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[16] 王红,朴范泽,侯喜林.牛呼吸道合胞体病毒RT-PCR检测方法的建立[J].动物医学进展,2008,11:1-4.
[17] 杨洺扬,王炜,李真光,等.牛呼吸道合胞体病检测方法研究进展[J].动物医学进展,2014(9):90-92.
[18] 霍烽,霍蕾.传染性鼻气管炎病几种检疫方法的比较[J].内蒙古农业大学学报,2006,27(1):141-143.
[2] PACCAUD M F,JACQUIER C.A respiratory syncytial virus of bovine origin[J].Archiv Fur Die Gesamte Virusforschung 1970,30(4):327-342.
[3] INABA Y,TANAKA Y,OMORI T,et al.Isolation of bovine respiratory syncytial virus[J].Tohoku J Exp Med,1970,40(6):473-474.
[4] SMITH M H,FREY M L,DIERKS R E.Isolation,characterization,and pathogenicity studies of a bovine respiratory syncytial virus[J].Arch Virol,1975,47:237-247.
[5] JACOBS J W,EDINGTON N.Isolation of respiratory syncytial virus from cattle in Britain[J].Vet Rec,1971,88(26):694.
[6] ELAZHARY M A,SILIM A,MORIN M.A natural outbreak of bovine respiratory disease caused by bovine respiratory syncytial virus[J].Cornell Vet,1982,72(3):325-333.
[7] BAKER J C,AMEs T R,MARKHAM R J.Serologic studies of bovine respiratory syncytial virus in Minnesota cattle[J].Am J Vet Res,1985,46(4):891-892.
[8] DUNCAN R B,POTGIETER L N.Antigenic diversity of respiratory syncytial viruses and its implications for prophylaxis in rumiants[J].Vet Microbiol,1993,37:319-341.
[9] 王丽荣.牛呼吸道合胞体病毒的病原学与诊治[J].黑龙江畜牧兽医,2006(6):70-71.
[10] LARSEN L E.Bovine respiratory syncytial virus (BRSV):a review[J].Acta Vet Scand,2000,41(1):1-24.
[11] BAKER J C,ELLIS J A,CLARK E G.Bovine respiratory syncytial virus[J].Vet Clin N Am Food A,1997,13(3):425-454.
[12] STOTT E J,TAYLOR G.Respiratory syncytial virus.brief review.archives[J].Virology,1985,84(1/2):1-52.
[13] 欧阳志明.牛呼吸道合胞体病毒感染[J].中国畜禽传染病,1995(1):62-63.
[14] 李丽阳,刘洋,王磊,等.脂筏在牛呼吸道合胞体病毒感染MDBK细胞中的作用[J].中国兽医学报,2017,37(4):643-647.
[15] BORSBERRY S.Challenges of BOHV-1 eradication in 'closed' cattle herds[J].Vet Rec,2012,170:652.
[16] 王红,朴范泽,侯喜林.牛呼吸道合胞体病毒RT-PCR检测方法的建立[J].动物医学进展,2008,11:1-4.
[17] 杨洺扬,王炜,李真光,等.牛呼吸道合胞体病检测方法研究进展[J].动物医学进展,2014(9):90-92.
[18] 霍烽,霍蕾.传染性鼻气管炎病几种检疫方法的比较[J].内蒙古农业大学学报,2006,27(1):141-143.