含4种食源性病毒检测靶标多联装甲RNA的制备、纯化与定值
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摘要
基于Qβ噬菌体装甲RNA制备平台构建同时含有诺如病毒(norovirus,NoV)、甲肝病毒(hepatitis Avirus,HAV)、轮状病毒(rotavirus,RV)、星状病毒(astrovirus,AstV)检测靶标RNA的多联装甲RNA(multiplexarmoredRNA,AR-MulV),并进行纯化与初步定值。结果表明,重组质粒在大肠杆菌中成功表达,表达产物大小约为14.1 kDa;制备的AR-MulV经纯化后无杂蛋白与残留重组质粒,电镜下可见大量结构形态完整的病毒样颗粒,大小约为25 nm。初步定值结果显示,AR-MulV中GⅠ型NoV、GⅡ型NoV、HAV、RV和AstV检测靶标RNA的含量分别为(1.24±0.2)×10~7、(2.54±0.6)×10~7、(2.24±0.3)×10~7、(2.96±0.5)×10~7 copies/μL和(3.19±0.4)×10~7 copies/μL。本研究基于Qβ噬菌体装甲RNA制备平台成功制备同时包含4种食源性病毒标准方法检测靶标的多联装甲RNA,为食源性病毒的分子检测以及多重实时荧光定量逆转录-聚合酶链式反应阳性质控样品的研发提供新思路。
This study was undertaken to construct multiplex armored RNA(AR-MulV) containing the target RNAs of norovirus(NoV), hepatitis A virus(HAV), rotavirus(RV), and astrovirus(AstV) based on Qβ bacteriophage for foodborne virus detection. The armored RNA was puri?ed and quanti?ed as well. The recombinant plasmid was ef?ciently expressed in Escherichia coli and the molecular mass of the product was estimated to be 14.1 kDa on SDS-PAGE. The puri?ed AR-MulV was found to be free of any unwanted protein or residual plasmid and it was observed as virus like particles with good structural and morphological integrity 25 nm in diameter under electron microscopy. The target RNAs of NoV(GⅠ and GⅡ), HAV, RV and AstV in the AR-MulV were quantified as(1.24 ± 0.2) × 10~7,(2.54 ± 0.6) × 10~7,(2.24 ± 0.3) ×10~7,(2.96 ± 0.5) × 10~7 and(3.19 ± 0.4) × 107 copies/μL respectively. The multiplex armored RNA based on Qβbacteriophage could serve as a good positive control material candidate for use in the simultaneous detection of 4 foodborne viruses. Moreover, this study provides a new strategy for preparing positive reference material for use in the development of a multiplex real-time RT-PCR assay for foodborne viruses.
This study was undertaken to construct multiplex armored RNA(AR-MulV) containing the target RNAs of norovirus(NoV), hepatitis A virus(HAV), rotavirus(RV), and astrovirus(AstV) based on Qβ bacteriophage for foodborne virus detection. The armored RNA was puri?ed and quanti?ed as well. The recombinant plasmid was ef?ciently expressed in Escherichia coli and the molecular mass of the product was estimated to be 14.1 kDa on SDS-PAGE. The puri?ed AR-MulV was found to be free of any unwanted protein or residual plasmid and it was observed as virus like particles with good structural and morphological integrity 25 nm in diameter under electron microscopy. The target RNAs of NoV(GⅠ and GⅡ), HAV, RV and AstV in the AR-MulV were quantified as(1.24 ± 0.2) × 10~7,(2.54 ± 0.6) × 10~7,(2.24 ± 0.3) ×10~7,(2.96 ± 0.5) × 10~7 and(3.19 ± 0.4) × 107 copies/μL respectively. The multiplex armored RNA based on Qβbacteriophage could serve as a good positive control material candidate for use in the simultaneous detection of 4 foodborne viruses. Moreover, this study provides a new strategy for preparing positive reference material for use in the development of a multiplex real-time RT-PCR assay for foodborne viruses.
引文
[1]LEE S Y,KIM M J,KIM H J,et al.Simultaneous detection of four foodborne viruses in food samples using a one-step multiplex reverse transcription PCR[J].Journal of Microbiology and Biotechnology,2018,28(2):210-217.DOI:10.4014/jmb.1710.10008.
[2]吴清平,寇晓霞,张菊梅.食源性病毒及其检测方法[J].微生物学通报,2004,31(3):101-105.DOI:10.3969/j.issn.0253-2654.2004.03.022.
[3]DESSELBERGER U.Rotaviruses[J].Virus Research,2014,190(7):75-96.DOI:10.1016/j.virusres.2014.06.016.
[4]BON F,FASCIA P,DAUVERGNE M,et al.Prevalence of group A rotavirus,human calicivirus,astrovirus,and adenovirus type40 and 41 infections among children with acute gastroenteritis in Dijon,France[J].Journal of Clinical Microbiology,1999,37(9):3055-3058.
[5]DENNEHY P H,NELSON S M,SPANGENBERGER S,et al.A prospective case-control study of the role of astrovirus in acute diarrhea among hospitalized young children[J].Journal of Infectious Diseases,2001,184(1):10-15.DOI:10.1086/321007.
[6]CLARK B,MCKENDRICK M.A review of viral gastroenteritis[J].Current Opinion in Infectious Diseases,2004,17(5):461-469.DOI:10.1097/00001432-200410000-00011.
[7]KOOPMANS M,DUIZER E.Foodborne viruses:an emerging problem[J].International Journal of Food Microbiology,2004,90(1):23-41.DOI:10.1016/S0168-1605(03)00169-7.
[8]王大勇,方振东,谢朝新,等.轮状病毒荧光定量PCR标准品的构建[J].后勤工程学院学报,2013,29(4):66-73.DOI:10.3969/j.issn.1672-7843.2013.04.012.
[9]徐蕾蕊,魏海燕,林长军,等.星状病毒核酸检测标准物质的研制[J].食品科学,2016,37(6):172-177.DOI:10.7506/spkx1002-6630-201606031.
[10]谷强,吴亚琼,高志强,等.西部马脑脊髓炎病毒实时荧光RT-PCR检测方法建立及标准质控样品制备[J].中国动物检疫,2013(11):80-84.DOI:10.3969/j.issn.1005-944X.2013.11.026.
[11]DUBOIS D B,MATTHEW M W,BRITTAN L P,et al.Ribonuclease resistant viral RNA standards:5677124[P].1997-10-14.DOI:US5677124A.
[12]SONG L Q,SUN S P,LI B,et al.External quality assessment for enterovirus 71 and coxsackievirus A16 detection by reverse transcription-PCR using armored RNA as a virus surrogate[J].Journal of Clinical Microbiology,2011,49(10):3591-3595.DOI:10.1128/JCM.00686-11.
[13]OKELLO J B A,RODRIGUEZ L,POINAR D,et al.Quantitative assessment of the sensitivity of various commercial reverse transcriptases based on armored HIV RNA[J].PLoS ONE,2013,5(11):e13931.DOI:10.1371/journal.pone.0013931.
[14]张奇,姚琳,江艳华,等.基于Qbeta噬菌体装甲RNA技术的诺如病毒RNA标准参考样品的研制[J].中国生物工程杂志,2018,38(1):42-50.DOI:10.13523/j.cb.20180105.
[15]吴亚琼,高志强,吴延功,等.亚洲I型口蹄疫病毒核酸检测标准物质的制备研究[J].中国兽医杂志,2012,48(8):8-12.DOI:10.3969/j.issn.0529-6005.2012.08.003.
[16]LIN G G,ZHANG K,DONG Z,et al.Fast preparation of a long chimeric armored RNA as controls for external quality assessment for molecular detection of Zika virus[J].Clinica Chimica Acta,2017,466:138-144.DOI:10.1016/j.cca.2017.01.023.
[17]WANG G J,ZHANG R,HAN Y X,et al.The evaluation of7 commercial real-time PCR kits for Zaire ebolavirus using virus-like particle-encapsulated EBOV RNA[J].Diagnostic Microbiology and Infectious Disease,2015,83(4):355-358.DOI:10.1016/j.diagmicrobio.2015.07.025.
[18]STEVENSON J,HYMAS W,HILLYARD D.The use of armored RNA as a multi-purpose internal control for RT-PCR[J].Journal of Virological Methods,2008,150(1/2):73-76.DOI:10.1016/j.jviromet.2008.02.007.
[19]ZHAN S,LI J M,XU R H,et al.Armored long RNA controls or standards for branched DNA assay for detection of human immunodeficiency virus type 1[J].Journal of Clinical Microbiology,2009,47(8):2571-2576.DOI:10.1128/JCM.00232-09.
[20]CHENG Y J,NIU J J,ZHANG Y Y,et al.Preparation of Histagged armored RNA phage particles as a control for real-time reverse transcription-PCR detection of severe acute respiratory syndrome coronavirus[J].Journal of Clinical Microbiology,2006,44(10):3557-3561.DOI:10.1128/JCM.00713-06.
[21]WEI Y X,YANG C M,WEI B K,et al.RNase-resistant viruslike particles containing long chimeric RNA sequences produced by two-plasmid coexpression system[J].Journal of Clinical Microbiology,2008,46(5):1734-1740.
[22]MONJURE C J,TATUM C D,PANGANIBAN A T,et al.Optimization of PCR for quantification of simian immunodeficiency virus genomic RNA in plasma of rhesus macaques(Macaca mulatta)using armored RNA[J].Journal of Medical Primatology,2014,43(1):31-43.DOI:10.1128/JCM.02248-07.
[23]张丹,聂凯,关丽,等.内含中东呼吸综合征冠状病毒部分N基因病毒样颗粒构建和表达[J].中华实验和临床病毒学杂志,2015,29(3):259-262.DOI:10.3760/cma.j.issn.1003-9279.2015.03.022.
[24]窦敏,张国广,于广福,等.含口蹄疫病毒IRES RNA病毒样颗粒表达载体的构建[J].中国生物工程杂志,2007,27(9):31-35.DOI:10.3969/j.issn.1671-8135.2007.09.006.
[25]乔彩霞,张鹤晓,高志强,等.内含猪繁殖与呼吸综合征病毒保守基因序列假病毒粒子的构建及应用[J].中国兽医学报,2016,36(12):2024-2029.DOI:10.16303/j.cnki.1005-4545.2016.12.07.
[26]DONIA D,DIVIZIA M,PANA’A.Use of armored RNA as a standard to construct a calibration curve for real-time RTPCR[J].Journal of Virological Methods,2005,126(1/2):157-163.DOI:10.1016/j.jviromet.2005.02.004.
[27]张国广,郭迟鸣,欧阳莹,等.病毒样颗粒为阳性对照的H5N1实时荧光定量PCR检测方法的建立[J].厦门大学学报(自然科学版),2014,53(3):418-423.DOI:10.6043/j.issn.0438-0479.2014.03.024.
[28]郑惠惠.中国部分甲肝病毒流行株结构蛋白VP3-VP1区基因特点分析及TaqMan Real-time RT-PCR法定量检测甲型肝炎病毒核酸[D].北京:中国疾病预防控制中心,2011.
[29]纪蕾,韩建康,吴晓芳,等.多重荧光RT-PCR同时检测GI型和GII型诺如病毒方法的建立[J].中国人兽共患病学报,2011,27(4):311-315.DOI:10.3969/j.issn.1002-2694.2011.04.011.
[30]KANG L H,OH S H,PARK J W,et al.Simultaneous detection of waterborne viruses by multiplex real-time PCR[J].Journal of Microbiology,2013,51(5):671-675.DOI:10.1007/s12275-013-3199-1.
[31]FUENTES C,GUI X S,PEREZ-RODRIGUEZ F J,et al.Standardized multiplex one-step qRT-PCR for hepatitis A virus,norovirus GIand GII quantification in bivalve mollusks and water[J].Food Microbiology,2014,40(1):55-63.DOI:10.1016/j.fm.2013.12.003.
[2]吴清平,寇晓霞,张菊梅.食源性病毒及其检测方法[J].微生物学通报,2004,31(3):101-105.DOI:10.3969/j.issn.0253-2654.2004.03.022.
[3]DESSELBERGER U.Rotaviruses[J].Virus Research,2014,190(7):75-96.DOI:10.1016/j.virusres.2014.06.016.
[4]BON F,FASCIA P,DAUVERGNE M,et al.Prevalence of group A rotavirus,human calicivirus,astrovirus,and adenovirus type40 and 41 infections among children with acute gastroenteritis in Dijon,France[J].Journal of Clinical Microbiology,1999,37(9):3055-3058.
[5]DENNEHY P H,NELSON S M,SPANGENBERGER S,et al.A prospective case-control study of the role of astrovirus in acute diarrhea among hospitalized young children[J].Journal of Infectious Diseases,2001,184(1):10-15.DOI:10.1086/321007.
[6]CLARK B,MCKENDRICK M.A review of viral gastroenteritis[J].Current Opinion in Infectious Diseases,2004,17(5):461-469.DOI:10.1097/00001432-200410000-00011.
[7]KOOPMANS M,DUIZER E.Foodborne viruses:an emerging problem[J].International Journal of Food Microbiology,2004,90(1):23-41.DOI:10.1016/S0168-1605(03)00169-7.
[8]王大勇,方振东,谢朝新,等.轮状病毒荧光定量PCR标准品的构建[J].后勤工程学院学报,2013,29(4):66-73.DOI:10.3969/j.issn.1672-7843.2013.04.012.
[9]徐蕾蕊,魏海燕,林长军,等.星状病毒核酸检测标准物质的研制[J].食品科学,2016,37(6):172-177.DOI:10.7506/spkx1002-6630-201606031.
[10]谷强,吴亚琼,高志强,等.西部马脑脊髓炎病毒实时荧光RT-PCR检测方法建立及标准质控样品制备[J].中国动物检疫,2013(11):80-84.DOI:10.3969/j.issn.1005-944X.2013.11.026.
[11]DUBOIS D B,MATTHEW M W,BRITTAN L P,et al.Ribonuclease resistant viral RNA standards:5677124[P].1997-10-14.DOI:US5677124A.
[12]SONG L Q,SUN S P,LI B,et al.External quality assessment for enterovirus 71 and coxsackievirus A16 detection by reverse transcription-PCR using armored RNA as a virus surrogate[J].Journal of Clinical Microbiology,2011,49(10):3591-3595.DOI:10.1128/JCM.00686-11.
[13]OKELLO J B A,RODRIGUEZ L,POINAR D,et al.Quantitative assessment of the sensitivity of various commercial reverse transcriptases based on armored HIV RNA[J].PLoS ONE,2013,5(11):e13931.DOI:10.1371/journal.pone.0013931.
[14]张奇,姚琳,江艳华,等.基于Qbeta噬菌体装甲RNA技术的诺如病毒RNA标准参考样品的研制[J].中国生物工程杂志,2018,38(1):42-50.DOI:10.13523/j.cb.20180105.
[15]吴亚琼,高志强,吴延功,等.亚洲I型口蹄疫病毒核酸检测标准物质的制备研究[J].中国兽医杂志,2012,48(8):8-12.DOI:10.3969/j.issn.0529-6005.2012.08.003.
[16]LIN G G,ZHANG K,DONG Z,et al.Fast preparation of a long chimeric armored RNA as controls for external quality assessment for molecular detection of Zika virus[J].Clinica Chimica Acta,2017,466:138-144.DOI:10.1016/j.cca.2017.01.023.
[17]WANG G J,ZHANG R,HAN Y X,et al.The evaluation of7 commercial real-time PCR kits for Zaire ebolavirus using virus-like particle-encapsulated EBOV RNA[J].Diagnostic Microbiology and Infectious Disease,2015,83(4):355-358.DOI:10.1016/j.diagmicrobio.2015.07.025.
[18]STEVENSON J,HYMAS W,HILLYARD D.The use of armored RNA as a multi-purpose internal control for RT-PCR[J].Journal of Virological Methods,2008,150(1/2):73-76.DOI:10.1016/j.jviromet.2008.02.007.
[19]ZHAN S,LI J M,XU R H,et al.Armored long RNA controls or standards for branched DNA assay for detection of human immunodeficiency virus type 1[J].Journal of Clinical Microbiology,2009,47(8):2571-2576.DOI:10.1128/JCM.00232-09.
[20]CHENG Y J,NIU J J,ZHANG Y Y,et al.Preparation of Histagged armored RNA phage particles as a control for real-time reverse transcription-PCR detection of severe acute respiratory syndrome coronavirus[J].Journal of Clinical Microbiology,2006,44(10):3557-3561.DOI:10.1128/JCM.00713-06.
[21]WEI Y X,YANG C M,WEI B K,et al.RNase-resistant viruslike particles containing long chimeric RNA sequences produced by two-plasmid coexpression system[J].Journal of Clinical Microbiology,2008,46(5):1734-1740.
[22]MONJURE C J,TATUM C D,PANGANIBAN A T,et al.Optimization of PCR for quantification of simian immunodeficiency virus genomic RNA in plasma of rhesus macaques(Macaca mulatta)using armored RNA[J].Journal of Medical Primatology,2014,43(1):31-43.DOI:10.1128/JCM.02248-07.
[23]张丹,聂凯,关丽,等.内含中东呼吸综合征冠状病毒部分N基因病毒样颗粒构建和表达[J].中华实验和临床病毒学杂志,2015,29(3):259-262.DOI:10.3760/cma.j.issn.1003-9279.2015.03.022.
[24]窦敏,张国广,于广福,等.含口蹄疫病毒IRES RNA病毒样颗粒表达载体的构建[J].中国生物工程杂志,2007,27(9):31-35.DOI:10.3969/j.issn.1671-8135.2007.09.006.
[25]乔彩霞,张鹤晓,高志强,等.内含猪繁殖与呼吸综合征病毒保守基因序列假病毒粒子的构建及应用[J].中国兽医学报,2016,36(12):2024-2029.DOI:10.16303/j.cnki.1005-4545.2016.12.07.
[26]DONIA D,DIVIZIA M,PANA’A.Use of armored RNA as a standard to construct a calibration curve for real-time RTPCR[J].Journal of Virological Methods,2005,126(1/2):157-163.DOI:10.1016/j.jviromet.2005.02.004.
[27]张国广,郭迟鸣,欧阳莹,等.病毒样颗粒为阳性对照的H5N1实时荧光定量PCR检测方法的建立[J].厦门大学学报(自然科学版),2014,53(3):418-423.DOI:10.6043/j.issn.0438-0479.2014.03.024.
[28]郑惠惠.中国部分甲肝病毒流行株结构蛋白VP3-VP1区基因特点分析及TaqMan Real-time RT-PCR法定量检测甲型肝炎病毒核酸[D].北京:中国疾病预防控制中心,2011.
[29]纪蕾,韩建康,吴晓芳,等.多重荧光RT-PCR同时检测GI型和GII型诺如病毒方法的建立[J].中国人兽共患病学报,2011,27(4):311-315.DOI:10.3969/j.issn.1002-2694.2011.04.011.
[30]KANG L H,OH S H,PARK J W,et al.Simultaneous detection of waterborne viruses by multiplex real-time PCR[J].Journal of Microbiology,2013,51(5):671-675.DOI:10.1007/s12275-013-3199-1.
[31]FUENTES C,GUI X S,PEREZ-RODRIGUEZ F J,et al.Standardized multiplex one-step qRT-PCR for hepatitis A virus,norovirus GIand GII quantification in bivalve mollusks and water[J].Food Microbiology,2014,40(1):55-63.DOI:10.1016/j.fm.2013.12.003.