口蹄疫病毒实时荧光RT-RPA快速检测方法的建立
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摘要
为建立一种简单、快速、特异的口蹄疫病毒(FMDV)分子检测方法,本研究基于FMDV的3D基因,设计特异性引物和exo探针,建立了用于FMDV快速检测的等温实时荧光反转录重组酶聚合酶(Real-time RT-RPA)方法。该方法采用便携式等温扩增仪-Genie Ⅲ实时监测扩增结果,40℃20 min即可完成检测。结果显示,FMDV RT-RPA方法能够特异性扩增FMDV 3D基因,而对水泡性口炎病毒、猪繁殖与呼吸综合征病毒、猪瘟病毒、脑心肌炎病毒等均无扩增;以体外转录的FMDV 3D RNA作为模板,该方法在95%置信区间检出下限为1.0×10~2拷贝/μL,组内和组间变异系数均小于1%;使用43份含有灭活FMDV的模拟临床样品分析显示,RT-RPA对FMDV的检出率为34.9%(15/43),略低于荧光定量RT-PCR的检出率(39.5%,17/43),而两种方法的符合率为95.3%(41/43)。RT-RPA能够在6 min～16 min内完成检测,而实时荧光RT-PCR则需要30 min～51 min。本研究所建立的实时荧光RT-RPA方法反应快速,特异性强,灵敏性高,操作简单,结合便携式具有荧光检测功能的等温扩增仪Genie Ⅲ,组建了FMDV快速检测平台,在基层兽医部门,尤其在野外及疫情现场的FMDV检测中具有极大的应用潜力,为设备仪器有限的实验室和田间对FMDV的快速检测提供了一种有效的方法,对于条件有限地区FMD的防控具有重要意义。
To develop a simple, rapid and specific method for the foot-and-mouth disease virus(FMDV) detection, the real-time reverse-transcription recombinase polymerase amplification assay(real-time RT-RPA) was developed using primers targetting the conserved region of the 3D gene, and the amplification was performed at 40℃ in the portable Genie Ⅲ scanner device for 20 min under the real-time monitoring. The amplification of real-time RT-RPA was specific for FMDV, but not for VSV,PRRSV, CSFV, EMCV, PRV and PCV2, displaying a high specificity. Using the in vitro transcribed FMDV RNA as template, the analytical sensitivity was 10~2 copies at 95% cases. The co-efficient of variations in intra-and inter-assays were both less than 1%.The assay performance was evaluated by testing 43 clinical samples by the real-time RT-RPA and a real-time RT-PCR assay. Fifteen samples were FMDV positive in RT-RPA(34.9%, 15/43), while the positive rate was 39.5%(17/43) in real-time RT-PCR. The diagnostic agreement between the two assays was 95.3%(41/43). It took 6-16 min in the RT-RPA assay to obtain the positive results, while the real-time RT-PCR took about 30 min to 51 min. The developed FMDV RT-RPA assay is rapid, highly specific,highly sensitive and easy to perform, and the FMDV rapid detection platform was established with Genie Ⅲ, which provides a rapid and reliable diagnostic tool for FMD in the field, and is of great significance in FMD provention.
To develop a simple, rapid and specific method for the foot-and-mouth disease virus(FMDV) detection, the real-time reverse-transcription recombinase polymerase amplification assay(real-time RT-RPA) was developed using primers targetting the conserved region of the 3D gene, and the amplification was performed at 40℃ in the portable Genie Ⅲ scanner device for 20 min under the real-time monitoring. The amplification of real-time RT-RPA was specific for FMDV, but not for VSV,PRRSV, CSFV, EMCV, PRV and PCV2, displaying a high specificity. Using the in vitro transcribed FMDV RNA as template, the analytical sensitivity was 10~2 copies at 95% cases. The co-efficient of variations in intra-and inter-assays were both less than 1%.The assay performance was evaluated by testing 43 clinical samples by the real-time RT-RPA and a real-time RT-PCR assay. Fifteen samples were FMDV positive in RT-RPA(34.9%, 15/43), while the positive rate was 39.5%(17/43) in real-time RT-PCR. The diagnostic agreement between the two assays was 95.3%(41/43). It took 6-16 min in the RT-RPA assay to obtain the positive results, while the real-time RT-PCR took about 30 min to 51 min. The developed FMDV RT-RPA assay is rapid, highly specific,highly sensitive and easy to perform, and the FMDV rapid detection platform was established with Genie Ⅲ, which provides a rapid and reliable diagnostic tool for FMD in the field, and is of great significance in FMD provention.
引文
[1]Alexandersen S,Zhang Z,Donaldson A I,et al.The pathogenesis and diagnosis of foot-and-mouth disease[J].J Comp Pathol,2003,129(1):1-36.
[2]何继军,郭建宏,刘湘涛.我国口蹄疫流行现状与控制策略[J].中国动物检疫,2015,(6):10-14.
[3]Knight-Jones T J D,Rushton J.The economic impacts of foot and mouth disease-What are they,how big are they and where do they occur?[J].Prev Vet Med,2013,112(3-4):161.
[4]Daher R K,Stewart G,Boissinot M,et al.Recombinase polymerase amplification for diagnostic applications[J].Clin Chem,2016,62(7):947-961.
[5]Piepenburg O,Williams C H,Stemple D L,et al.DNA detection using recombination proteins[J].PLos Biol,2006,4(7):1115-1121.
[6]Wang Jian-chang,Wang Jin-feng,Liu Li-bing,et al.Development of a real-time recombinase polymerase amplification assay for rapid and sensitive detection of porcine circovirus 2[J].Arch Virol,2017,162(8):2293-2296.
[7]Wang Jian-chang,Liu Li-bing,Han Qing-an,et al.An exo probe-based recombinase polymerase amplification assay for the rapid detection of porcine parvovirus[J].J Virol Methods,2017,248(7):145-147.
[8]Abd El Wahed A,El-Deeb A,El-Tholoth M,et al.A portable reverse transcription recombinase polymerase amplification assay for rapid detection of foot-and-mouth disease virus[J].PLoSOne,2013,8(8):e71642.
[9]杨洋,秦晓东,宋一鸣,等.口蹄疫病毒real-time RT-RPA检测方法的建立与应用[J].黑龙江畜牧兽医,2017,11(9):172-176.
[10]Callahan J D,Brown F,Osorio F A,et al.Use of a portable real-time reverse transcriptasepolymerase chain reaction assay for rapid detection of foot-and-mouth disease virus[J].J Am Vet Med Assoc,2002,220(11):1636-1642.
[11]Ferris N P,Nordengrahn A,Hutchings G H,et al.Development and laboratory validation of a lateral flow device for the detection of foot-and-mouth disease virus in clinical samples[J].JVirol Methods,2009,155(1):10-17.
[12]Jamal S M,Belsham G J.Foot-and-mouth disease:past,present and future[J].Vet Res,2013,44(1):116-119.
[13]Shaw A E,Reid S M,Ebert K,et al.Implementation of a one-step real-time RT-PCR protocol for diagnosis of foot-andmouth disease[J].J Virol Methods,2007,143(1):81-85.
[14]Ambagala A,Fisher M,Goolia M,et al.Field-deployable reverse transcription-insulated isothermal PCR(RT-iiPCR)assay for rapid and sensitive detection of Foot-and-mouth disease virus[J].Transbound Emerg Dis,2016,64(5):1610-1623.
[15]Dukes J P,King D P,Alexandersen S.Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus[J].Arch Virol,2006,151(6):1093-1106.
[16]金静维,马保华,邱索平,等.口蹄疫病毒解旋酶恒温扩增检测方法的建立[J].贵州畜牧兽医,2014,(5):1-5.
[2]何继军,郭建宏,刘湘涛.我国口蹄疫流行现状与控制策略[J].中国动物检疫,2015,(6):10-14.
[3]Knight-Jones T J D,Rushton J.The economic impacts of foot and mouth disease-What are they,how big are they and where do they occur?[J].Prev Vet Med,2013,112(3-4):161.
[4]Daher R K,Stewart G,Boissinot M,et al.Recombinase polymerase amplification for diagnostic applications[J].Clin Chem,2016,62(7):947-961.
[5]Piepenburg O,Williams C H,Stemple D L,et al.DNA detection using recombination proteins[J].PLos Biol,2006,4(7):1115-1121.
[6]Wang Jian-chang,Wang Jin-feng,Liu Li-bing,et al.Development of a real-time recombinase polymerase amplification assay for rapid and sensitive detection of porcine circovirus 2[J].Arch Virol,2017,162(8):2293-2296.
[7]Wang Jian-chang,Liu Li-bing,Han Qing-an,et al.An exo probe-based recombinase polymerase amplification assay for the rapid detection of porcine parvovirus[J].J Virol Methods,2017,248(7):145-147.
[8]Abd El Wahed A,El-Deeb A,El-Tholoth M,et al.A portable reverse transcription recombinase polymerase amplification assay for rapid detection of foot-and-mouth disease virus[J].PLoSOne,2013,8(8):e71642.
[9]杨洋,秦晓东,宋一鸣,等.口蹄疫病毒real-time RT-RPA检测方法的建立与应用[J].黑龙江畜牧兽医,2017,11(9):172-176.
[10]Callahan J D,Brown F,Osorio F A,et al.Use of a portable real-time reverse transcriptasepolymerase chain reaction assay for rapid detection of foot-and-mouth disease virus[J].J Am Vet Med Assoc,2002,220(11):1636-1642.
[11]Ferris N P,Nordengrahn A,Hutchings G H,et al.Development and laboratory validation of a lateral flow device for the detection of foot-and-mouth disease virus in clinical samples[J].JVirol Methods,2009,155(1):10-17.
[12]Jamal S M,Belsham G J.Foot-and-mouth disease:past,present and future[J].Vet Res,2013,44(1):116-119.
[13]Shaw A E,Reid S M,Ebert K,et al.Implementation of a one-step real-time RT-PCR protocol for diagnosis of foot-andmouth disease[J].J Virol Methods,2007,143(1):81-85.
[14]Ambagala A,Fisher M,Goolia M,et al.Field-deployable reverse transcription-insulated isothermal PCR(RT-iiPCR)assay for rapid and sensitive detection of Foot-and-mouth disease virus[J].Transbound Emerg Dis,2016,64(5):1610-1623.
[15]Dukes J P,King D P,Alexandersen S.Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus[J].Arch Virol,2006,151(6):1093-1106.
[16]金静维,马保华,邱索平,等.口蹄疫病毒解旋酶恒温扩增检测方法的建立[J].贵州畜牧兽医,2014,(5):1-5.