树鼩粪便中病毒三种浓缩方法的效果比较
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摘要
目的比较树鼩粪便中病毒的三种浓缩方法,评价各方法的浓缩效果,为病毒浓缩方法的最佳选择提供依据。方法将指示病毒(树鼩呼肠孤病毒,tree shrew’s reovirus,TRV)加入经离心过滤处理后的野生树鼩粪便上清中,采用PEG(聚乙二醇,polyehhylene glycol)沉淀结合超声分离法、铁离子絮凝的化学方法和GBviroFast病毒浓缩试剂盒对树鼩粪便上清分别进行浓缩,经实时荧光定量PCR技术对指示病毒进行绝对定量,比较各方法的浓缩效果。结果 TRV病毒悬液的载量为1.34E+05;铁离子絮凝的化学方法浓缩的TRV载量为1.86E+04,浓缩效率为49.57%;GBviroFast病毒浓缩试剂盒浓缩的TRV载量为0.98E+04,浓缩效率为26.12%;PEG沉淀结合超声分离法浓缩的TRV载量为2.30E+03,浓缩效率为6.13%。结论三种方法均能浓缩粪便上清中低浓度的肠道病毒,铁离子絮凝方法最佳,GBviroFast病毒浓缩试剂盒其次,PEG结合超声分离效果最差。
Objective To provide a simple and efficient method for concentration of the virus in feces of Tupaia belangeri,the sensitivity,different concentrations and recovery of three method for enterovirus concentration were observed. Method The centrifuged and filtered wild tree shrews stool supernatant was added indicative virus(tree shrew's reovirus TRV),and the fecal supernatants were concentrated by PEG precipitation combined with ultrasonic separation,Fe~(3+)-based virus occulation and GBviro Fast concentrated virus kit. To determine the effect of concentrating three method,real-time PCR technique was used. Result Load of TRV virus suspension is1. 34 E + 05; TRV load concentrated by Fe~(3+)-based chemical flocculation is 1. 86 E + 04,and the concentration efficiency is 49. 57%. TRV load concentrated by GBviro Fast concentrated virus kit is 0. 98 E + 04,with concentrated efficiency of 26. 12%. TRV load concentrated by PEG precipitation combined with ultrasonic separation is 2. 30 E + 03,and the concentration efficiency only is 6. 13%. Conclusion Three method can concentrate stool supernatant which contain low concentrations of enteroviruses,Fe-based chemical flocculation is the best,GBviro Fast concentrated virus kit secondly,PEG combined with ultrasonic separation method is the worst.
Objective To provide a simple and efficient method for concentration of the virus in feces of Tupaia belangeri,the sensitivity,different concentrations and recovery of three method for enterovirus concentration were observed. Method The centrifuged and filtered wild tree shrews stool supernatant was added indicative virus(tree shrew's reovirus TRV),and the fecal supernatants were concentrated by PEG precipitation combined with ultrasonic separation,Fe~(3+)-based virus occulation and GBviro Fast concentrated virus kit. To determine the effect of concentrating three method,real-time PCR technique was used. Result Load of TRV virus suspension is1. 34 E + 05; TRV load concentrated by Fe~(3+)-based chemical flocculation is 1. 86 E + 04,and the concentration efficiency is 49. 57%. TRV load concentrated by GBviro Fast concentrated virus kit is 0. 98 E + 04,with concentrated efficiency of 26. 12%. TRV load concentrated by PEG precipitation combined with ultrasonic separation is 2. 30 E + 03,and the concentration efficiency only is 6. 13%. Conclusion Three method can concentrate stool supernatant which contain low concentrations of enteroviruses,Fe-based chemical flocculation is the best,GBviro Fast concentrated virus kit secondly,PEG combined with ultrasonic separation method is the worst.
引文
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[11]赵霖,周文亭,曹经瑗,等.模拟水样中甲肝病毒超滤浓缩和PEG浓缩方法的比较[J].中华实验和临床病毒学杂志,2016,(1):71-75
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[13]鲍琳琳,邓巍,孙丽华,等.AT-2灭活HIV-1病毒及纯化回收鉴定[J].中国比较医学杂志,2011,21(4):21-23.
[14]Hata A,Matsumori K,Kitajima M,et al.Concentration of Enteric Viruses in Large Volumes of Water Using a CartridgeType Mixed Cellulose Ester Membrane[J].Food&Environmental Virology,2015,7(1):1-7.
[15]李娟,黄健,唐学玺.一种从大体积海水中有效提取病毒颗粒的简易方法[J].渔业科学进展,2005,26(2):68-72.
[16]刘军义,吴清平,盘宝进,等.水体病毒钙离子絮凝浓缩新方法研究[J].微生物学通报,2007,34(6):1202-1204.
[17]Hurwitz B L,Li D,Poulos B T,et al.Evaluation of methods to concentrate and purify ocean virus communities through comparative,replicated metagenomics[J].Environmental Microbiology,2013,15(5):1428-1440.
[18]韩传银,张飞雄,童贻刚.噬菌体浓缩方法的比较[J].生物技术通讯,2013(5):695-697.
[19]蔡杰,许纪玲,陆玉婷,等.大容量样本病毒浓缩HCV核酸检测方案的研究[J].临床和实验医学杂志,2014,(13):1070-1073.
[20]Colombet J,Robin A,Lavie L,et al.Virioplankton‘pegylation’:Use of PEG(polyethylene glycol)to concentrate and purify viruses in pelagic ecosystems[J].Journal of Microbiological Methods,2007,71(3):212-219.
[2]Borchardt M A,Bertz P D,Spencer S K,et al.Incidence of Enteric Viruses in Groundwater from Household Wells in Wisconsin[J].Applied&Environmental Microbiology,2003,69(2):1172-1180.
[3]Kim S H,Cheon D S,Kim J H,et al.Outbreaks of gastroenteritis that occurred during school excursions in Korea were associated with several waterborne strains of norovirus[J].Journal of Clinical Microbiology,2005,43(9):4836-4839.
[4]Rutjes S,Italiaander R,van den Berg H H,et al.Isolation and detection of enterovirus RNA from large-volume water samples by using the Nucli Sens mini MAG system and real-time nucleic acid sequence-based amplification[J].Appl Environ Microbiol,2005,71(7):3734-3740.
[5]Shah J D,Baller J,Ying Z,et al.Comparison of tissue sample processing methods for harvesting the viral metagenome and a snapshot of the RNA viral community in a turkey gut[J].Journal of Virological Methods,2014,209:15-24.
[6]John S G,Mendez C B,Li D,et al.A simple and efficient method for concentration of ocean viruses by chemical flocculation[J].Environmental Microbiology Reports,2010,3(2):195-202.
[7]寇晓霞,吴清平,姚琳,等.水体中病毒浓缩方法及其条件优化[J].微生物学通报,2009,36(1):25-30.
[8]徐露,李金娟,欧阳雅博,等.水体中病毒的三种浓缩方法及其效果比较[J].环境与健康杂志,2011,28(2):108-110.
[9]李晓飞,殷安国,张媛,等.树鼩呼肠孤病毒RT-n PCR检测方法的建立及初步应用[J].中国比较医学杂志,2014(6):63-68.
[10]Kunze A,Lu P,Elssser D,et al.High performance concentration method for viruses in drinking water[J].Journal of Virological Methods,2015,222:132-137.
[11]赵霖,周文亭,曹经瑗,等.模拟水样中甲肝病毒超滤浓缩和PEG浓缩方法的比较[J].中华实验和临床病毒学杂志,2016,(1):71-75
[12]杜丽娟,王世光,张立,等.两种方法纯化流感病毒的比较[J].中国生物制品学杂志,2007,20(3):216-218.
[13]鲍琳琳,邓巍,孙丽华,等.AT-2灭活HIV-1病毒及纯化回收鉴定[J].中国比较医学杂志,2011,21(4):21-23.
[14]Hata A,Matsumori K,Kitajima M,et al.Concentration of Enteric Viruses in Large Volumes of Water Using a CartridgeType Mixed Cellulose Ester Membrane[J].Food&Environmental Virology,2015,7(1):1-7.
[15]李娟,黄健,唐学玺.一种从大体积海水中有效提取病毒颗粒的简易方法[J].渔业科学进展,2005,26(2):68-72.
[16]刘军义,吴清平,盘宝进,等.水体病毒钙离子絮凝浓缩新方法研究[J].微生物学通报,2007,34(6):1202-1204.
[17]Hurwitz B L,Li D,Poulos B T,et al.Evaluation of methods to concentrate and purify ocean virus communities through comparative,replicated metagenomics[J].Environmental Microbiology,2013,15(5):1428-1440.
[18]韩传银,张飞雄,童贻刚.噬菌体浓缩方法的比较[J].生物技术通讯,2013(5):695-697.
[19]蔡杰,许纪玲,陆玉婷,等.大容量样本病毒浓缩HCV核酸检测方案的研究[J].临床和实验医学杂志,2014,(13):1070-1073.
[20]Colombet J,Robin A,Lavie L,et al.Virioplankton‘pegylation’:Use of PEG(polyethylene glycol)to concentrate and purify viruses in pelagic ecosystems[J].Journal of Microbiological Methods,2007,71(3):212-219.