埃博拉病毒扎伊尔亚型多引物自配引发等温扩增方法的建立
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摘要
鉴于目前埃博拉疫情在西非流行和输入国内的潜在风险,开发快速准确易普及的检测埃博拉病毒的方法对埃博拉防控具有重大意义。本文拟建立一种基于颜色判定简单、快速和灵敏的方法,即多引物自配引发等温扩增技术(isothermal multiple self-matching initiated amplification,IMSA)应用于埃博拉病毒扎伊尔亚型的检测。该技术设计了分别对应于埃博拉病毒扎伊尔型核蛋白(nuclear protein,NP)基因的7个区段的6条特异引物,在等温(63℃)条件下进行核酸扩增反应1小时,在扩增前加入HNB染料(羟基萘酚蓝)作为反应指示剂,以HNB染料颜色变化作为结果判定的标准。文中利用这种技术与RT-qPCR分别对含有目的片段的假病毒的系列稀释物以及模拟临床样本进行灵敏度分析,同时对30例健康人血浆以及登革病毒、日本脑炎病毒样本进行特异性分析。在塞拉利昂完成了81例埃博拉疑似病例血液样本的检测。结果显示RT-IMSA方法检测灵敏度与RT-qPCR方法的灵敏度相当,30例健康人血浆样本检测均为阴性,并且与登革病毒及日本脑炎病毒无交叉反应。与获得批准的荧光定量PCR试剂盒比较,RT-IMSA检测81例临床样本的结果为53例阳性,23例阴性,5例假阴性。灵敏度和特异性分别为91.4%和100%。因此,相较于RT-qPCR的高成本和复杂操作,RT-IMSA方法具有快速、简单的检测优势。
Given the Ebola outbreak in West Africa and the risks of spread to other regions,a rapid,sensitive and simple method for the detection of the Ebola virus(EBOV)is of great significance for the prevention and control of Ebola.We developed a simple colorimetric isothermal multiple self-matching initiated amplification(IMSA)for rapid detection of the Zaire subtype of the Ebola virus(EBOV-Z).This method employed six primers that recognized seven sites of the EBOV-Z nucleoprotein gene for amplification of nucleic acids under isothermal conditions at 63℃for 1h.Amplification products were detected through visual inspection of color change by pre-addition of hydroxyl naphthol blue dye.Relative sensitivity was validated by detection of serial tenfold dilutions of virus-like particles containing the partial EBOV-Z nucleoprotein gene and mock clinical sample.Specificity of IMSA was validated by detection of the plasma of 30 healthy volunteers,the dengue virus,and Japanese encephalitis virus.IMSA had comparable sensitivity to Reverse transcription-quantitative polymerase chain reaction(RT-qPCR),and cross-reaction with human plasma or other viruses was not observed.Reverse transcription-isothermal multiple self-matching initiated amplification(RT-IMSA)was also evaluated and compared in parallel with the commercial RT-qPCR kit for detection of EBOV-suspected samples of human blood in Sierra Leone.Sensitivity and specificity of the RT-IMSA was 91.4%and 100%,respectively.These data suggest that RT-IMSA is a valuable tool for the detection of the EBOV with the distinct advantages of simplicity and low cost compared with RT-qPCR.
Given the Ebola outbreak in West Africa and the risks of spread to other regions,a rapid,sensitive and simple method for the detection of the Ebola virus(EBOV)is of great significance for the prevention and control of Ebola.We developed a simple colorimetric isothermal multiple self-matching initiated amplification(IMSA)for rapid detection of the Zaire subtype of the Ebola virus(EBOV-Z).This method employed six primers that recognized seven sites of the EBOV-Z nucleoprotein gene for amplification of nucleic acids under isothermal conditions at 63℃for 1h.Amplification products were detected through visual inspection of color change by pre-addition of hydroxyl naphthol blue dye.Relative sensitivity was validated by detection of serial tenfold dilutions of virus-like particles containing the partial EBOV-Z nucleoprotein gene and mock clinical sample.Specificity of IMSA was validated by detection of the plasma of 30 healthy volunteers,the dengue virus,and Japanese encephalitis virus.IMSA had comparable sensitivity to Reverse transcription-quantitative polymerase chain reaction(RT-qPCR),and cross-reaction with human plasma or other viruses was not observed.Reverse transcription-isothermal multiple self-matching initiated amplification(RT-IMSA)was also evaluated and compared in parallel with the commercial RT-qPCR kit for detection of EBOV-suspected samples of human blood in Sierra Leone.Sensitivity and specificity of the RT-IMSA was 91.4%and 100%,respectively.These data suggest that RT-IMSA is a valuable tool for the detection of the EBOV with the distinct advantages of simplicity and low cost compared with RT-qPCR.
引文
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[9]Ksiazek T G,Rollin P E,Jahrling P B,Johnson E,Dalgard D W,Peters C J.Enzyme immunosorbent assay for Ebola virus antigens in tissues of infected primates[J].J Clin Microbiol,1992,30(4):947-950.
[10]Saijo M,Niikura M,Morikawa S,Ksiazek T G,Meyer R F,Peters C J,Kurane I.Enzyme-linked immunosorbent assays for detection of antibodies to Ebola and Marburg viruses using recombinant nucleoproteins[J].J Clin Microbiol,2001,39(1):1-7.
[11]Saijo M,Niikura M,Morikawa S,Kurane I.Immunofluorescence method for detection of Ebola virus immunoglobulin g,using HeLa cells which express recombinant nucleoprotein[J].J Clin Microbiol,2001,39(2):776-778.
[12]Sanchez A,Ksiazek T G,Rollin P E,Miranda M E,Trappier S G,Khan A S,Peters C J,Nichol S T.Detection and molecular characterization of Ebola viruses causing disease in human and nonhuman primates[J].J Infect Dis,1999,179Suppl 1:S164-169.
[13]Stephens K W,Hutchins R J,Dauphin L A.Crossplatform evaluation of commercial real-time reverse transcription PCR master mix kits using aquantitative5′nuclease assay for Ebola virus[J].Mol Cell Probes,2010,24(6):370-375.
[14]Towner J S,Sealy T K,Ksiazek T G,Nichol S T.High-throughput molecular detection of hemorrhagic fever virus threats with applications for outbreak settings[J].J Infect Dis,2007,196Suppl 2:S205-212.
[15]Trombley A R,Wachter L,Garrison J,Buckley-Beason V A,Jahrling J,Hensley L E,Schoepp R J,Norwood D A,Goba A,Fair J N,Kulesh D A.Comprehensive panel of real-time TaqMan polymerase chain reaction assays for detection and absolute quantification of filoviruses,arenaviruses,and New World hantaviruses[J].Am J Trop Med Hyg,2010,82(5):954-960.
[16]Towner J S,Rollin P E,Bausch D G,Sanchez A,Crary S M,Vincent M,Lee W F,Spiropoulou C F,Ksiazek T G,Lukwiya M,Kaducu F,Downing R,Nichol S T.Rapid Diagnosis of Ebola Hemorrhagic Fever by Reverse Transcription-PCR in an Outbreak Setting and Assessment of Patient Viral Load as a Predictor of Outcome[J].Journal of Virology,2004,78(8):4330-4341.
[17]Weidmann M,Muhlberger E,Hufert F T.Rapid detection protocol for filoviruses[J].J Clin Virol,2004,30(1):94-99.
[18]Ding X,Nie K,Shi L,Zhang Y,Guan L,Zhang D,Qi S,Ma X.Improved detection limit in rapid detection of human enterovirus 71and coxsackievirus A16by a novel reverse transcription-isothermal multipleself-matching-initiated amplification assay[J].J Clin Microbiol,2014,52(6):1862-1870.
[19]李阿茜,李伟红,李建东,张硕,曲靖,李川,张全福,梁米芳,李德新.扎伊尔型埃博拉病毒的实时荧光RTPCR检测方法研究[J].中华实验和临床病毒学杂志,2014,28(5):2.
[20]Ogawa H,Miyamoto H,Ebihara H,Ito K,Morikawa S,Feldmann H,Takada A.Detection of all known filovirus species by reverse transcription-polymerase chain reaction using aprimer set specific for the viral nucleoprotein gene[J].J Virol Methods,2011,171(1):310-313.
[21]Gibb T R,Norwood D A,Jr.,Woollen N,Henchal E A.Development and evaluation of a fluorogenic 5′nuclease assay to detect and differentiate between Ebola virus subtypes Zaire and Sudan[J].J Clin Microbiol,2001,39(11):4125-4130.
[22]Kurosaki Y,Takada A,Ebihara H,Grolla A,Kamo N,Feldmann H,Kawaoka Y,Yasuda J.Rapid and simple detection of Ebola virus by reverse transcriptionloop-mediated isothermal amplification[J].J Virol Methods,2007,141(1):78-83.
[23]Goldmeyer J,Kong H,Tang W.Development of a novel one-tube isothermal reverse transcription thermophilic helicase-dependent amplification platform for rapid RNA detection[J].J Mol Diagn,2007,9(5):639-644.
[24]Drosten C,Gottig S,Schilling S,Asper M,Panning M,Schmitz H,Gunther S.Rapid detection and quantification of RNA of Ebola and Marburg viruses,Lassa virus,Crimean-Congo hemorrhagic fever virus,Rift Valley fever virus,dengue virus,and yellow fever virus by real-time reverse transcription-PCR[J].J Clin Microbiol,2002,40(7):2323-2330.
[25]Liu L,Sun Y,Kargbo B,Zhang C,Feng H,Lu H,Liu W,Wang C,Hu Y,Deng Y,Jiang J,Kang X,Yang H,Jiang Y,Yang Y,Kargbo D,Qian J,Chen W.Detection of Zaire Ebola virus by real-time reverse transcription-polymerase chain reaction,Sierra Leone,2014[J].J Virol Methods,2015,222:62-65.
[2]Gire S K,Goba A,Andersen K G,Sealfon R S,Park D J,Kanneh L,Jalloh S,Momoh M,Fullah M,Dudas G,Wohl S,Moses L M,Yozwiak N L,Winnicki S,Matranga C B,Malboeuf C M,Qu J,Gladden A D,Schaffner S F,Yang X,Jiang P P,Nekoui M,Colubri A,Coomber M R,Fonnie M,Moigboi A,Gbakie M,Kamara F K,Tucker V,Konuwa E,Saffa S,Sellu J,Jalloh A A,Kovoma A,Koninga J,Mustapha I,Kargbo K,Foday M,Yillah M,Kanneh F,Robert W,Massally J L,Chapman S B,Bochicchio J,Murphy C,Nusbaum C,Young S,Birren B W,Grant D S,Scheiffelin J S,Lander E S,Happi C,Gevao S M,Gnirke A,Rambaut A,Garry R F,Khan S H,Sabeti P C.Genomic surveillance elucidates Ebola virus origin and transmission during the 2014outbreak[J].Science,2014,345(6202):1369-1372.
[3]Gatherer D.The 2014Ebola virus disease outbreak in West Africa[J].J Gen Virol,2014,95(Pt 8):1619-1624.
[4]World Health Organization.Ebola Situation Report-7October 2015.2015-10-14;http://apps.who.int/ebola/current-situation/ebola-situation-report-7-october-2015.
[5]Barrette R W,Metwally S A,Rowland J M,Xu L,Zaki S R,Nichol S T,Rollin P E,Towner J S,Shieh W J,Batten B,Sealy T K,Carrillo C,Moran K E,Bracht A J,Mayr G A,Sirios-Cruz M,Catbagan D P,Lautner E A,Ksiazek T G,White W R,McIntosh M T.Discovery of swine as a host for the Reston ebolavirus[J].Science,2009,325(5937):204-206.
[6]Towner J S,Sealy T K,Khristova M L,Albarino C G,Conlan S,Reeder S A,Quan P L,Lipkin W I,Downing R,Tappero J W,Okware S,Lutwama J,Bakamutumaho B,Kayiwa J,Comer J A,Rollin P E,Ksiazek T G,Nichol S T.Newly discovered ebola virus associated with hemorrhagic fever outbreak in Uganda[J/OL].PLoS Pathog,2008,4(11):e1000212.
[7]Rodriguez L L,De Roo A,Guimard Y,Trappier S G,Sanchez A,Bressler D,Williams A J,Rowe A K,Bertolli J,Khan A S,Ksiazek T G,Peters C J,and Nichol S T.Persistence genetic stability of Ebola virus during the outbreak in Kikwit,Democratic Republic of the Congo,1995[J].J Infect Dis,1999,179Suppl 1:S170-176.
[8]Geisbert T W,Jahrling P B.Differentiation of filoviruses by electron microscopy[J].Virus Res,1995,39(2-3):129-150.
[9]Ksiazek T G,Rollin P E,Jahrling P B,Johnson E,Dalgard D W,Peters C J.Enzyme immunosorbent assay for Ebola virus antigens in tissues of infected primates[J].J Clin Microbiol,1992,30(4):947-950.
[10]Saijo M,Niikura M,Morikawa S,Ksiazek T G,Meyer R F,Peters C J,Kurane I.Enzyme-linked immunosorbent assays for detection of antibodies to Ebola and Marburg viruses using recombinant nucleoproteins[J].J Clin Microbiol,2001,39(1):1-7.
[11]Saijo M,Niikura M,Morikawa S,Kurane I.Immunofluorescence method for detection of Ebola virus immunoglobulin g,using HeLa cells which express recombinant nucleoprotein[J].J Clin Microbiol,2001,39(2):776-778.
[12]Sanchez A,Ksiazek T G,Rollin P E,Miranda M E,Trappier S G,Khan A S,Peters C J,Nichol S T.Detection and molecular characterization of Ebola viruses causing disease in human and nonhuman primates[J].J Infect Dis,1999,179Suppl 1:S164-169.
[13]Stephens K W,Hutchins R J,Dauphin L A.Crossplatform evaluation of commercial real-time reverse transcription PCR master mix kits using aquantitative5′nuclease assay for Ebola virus[J].Mol Cell Probes,2010,24(6):370-375.
[14]Towner J S,Sealy T K,Ksiazek T G,Nichol S T.High-throughput molecular detection of hemorrhagic fever virus threats with applications for outbreak settings[J].J Infect Dis,2007,196Suppl 2:S205-212.
[15]Trombley A R,Wachter L,Garrison J,Buckley-Beason V A,Jahrling J,Hensley L E,Schoepp R J,Norwood D A,Goba A,Fair J N,Kulesh D A.Comprehensive panel of real-time TaqMan polymerase chain reaction assays for detection and absolute quantification of filoviruses,arenaviruses,and New World hantaviruses[J].Am J Trop Med Hyg,2010,82(5):954-960.
[16]Towner J S,Rollin P E,Bausch D G,Sanchez A,Crary S M,Vincent M,Lee W F,Spiropoulou C F,Ksiazek T G,Lukwiya M,Kaducu F,Downing R,Nichol S T.Rapid Diagnosis of Ebola Hemorrhagic Fever by Reverse Transcription-PCR in an Outbreak Setting and Assessment of Patient Viral Load as a Predictor of Outcome[J].Journal of Virology,2004,78(8):4330-4341.
[17]Weidmann M,Muhlberger E,Hufert F T.Rapid detection protocol for filoviruses[J].J Clin Virol,2004,30(1):94-99.
[18]Ding X,Nie K,Shi L,Zhang Y,Guan L,Zhang D,Qi S,Ma X.Improved detection limit in rapid detection of human enterovirus 71and coxsackievirus A16by a novel reverse transcription-isothermal multipleself-matching-initiated amplification assay[J].J Clin Microbiol,2014,52(6):1862-1870.
[19]李阿茜,李伟红,李建东,张硕,曲靖,李川,张全福,梁米芳,李德新.扎伊尔型埃博拉病毒的实时荧光RTPCR检测方法研究[J].中华实验和临床病毒学杂志,2014,28(5):2.
[20]Ogawa H,Miyamoto H,Ebihara H,Ito K,Morikawa S,Feldmann H,Takada A.Detection of all known filovirus species by reverse transcription-polymerase chain reaction using aprimer set specific for the viral nucleoprotein gene[J].J Virol Methods,2011,171(1):310-313.
[21]Gibb T R,Norwood D A,Jr.,Woollen N,Henchal E A.Development and evaluation of a fluorogenic 5′nuclease assay to detect and differentiate between Ebola virus subtypes Zaire and Sudan[J].J Clin Microbiol,2001,39(11):4125-4130.
[22]Kurosaki Y,Takada A,Ebihara H,Grolla A,Kamo N,Feldmann H,Kawaoka Y,Yasuda J.Rapid and simple detection of Ebola virus by reverse transcriptionloop-mediated isothermal amplification[J].J Virol Methods,2007,141(1):78-83.
[23]Goldmeyer J,Kong H,Tang W.Development of a novel one-tube isothermal reverse transcription thermophilic helicase-dependent amplification platform for rapid RNA detection[J].J Mol Diagn,2007,9(5):639-644.
[24]Drosten C,Gottig S,Schilling S,Asper M,Panning M,Schmitz H,Gunther S.Rapid detection and quantification of RNA of Ebola and Marburg viruses,Lassa virus,Crimean-Congo hemorrhagic fever virus,Rift Valley fever virus,dengue virus,and yellow fever virus by real-time reverse transcription-PCR[J].J Clin Microbiol,2002,40(7):2323-2330.
[25]Liu L,Sun Y,Kargbo B,Zhang C,Feng H,Lu H,Liu W,Wang C,Hu Y,Deng Y,Jiang J,Kang X,Yang H,Jiang Y,Yang Y,Kargbo D,Qian J,Chen W.Detection of Zaire Ebola virus by real-time reverse transcription-polymerase chain reaction,Sierra Leone,2014[J].J Virol Methods,2015,222:62-65.