摘要
目前,由于水环境污染或水系传播引起的病原体感染疾病是世界危害范围最广的问题,引发了许多流行事件,尤其是肠道病毒,引发的案例最多且流行面积最广。因此,对于肠道病毒的研究也就成为当前人们研究的热点问题之一。然而,水体中肠道病毒的含量较少,浓度很低,无法进行直接检测,必须首先从水体中浓缩。另外,传统的细胞培养检测方法,操作繁琐,耗时长,难以在基础实验室进行普及推广。因此,选择一种高效的浓缩方法和快速灵敏的检测方法对环境水体中病毒研究具有很重要的现实意义。
本次研究对国内外现有的各种浓缩方法进行了综述比较,并选择了其中的微孔滤膜吸附-洗脱法、滑石粉-硅藻土吸附层吸附-洗脱法、化学絮凝法和滑石粉-硅藻土絮凝沉淀法四种不同的浓缩方法进行比较研究,综合考虑病毒回收率、可靠性等各个方面的因素,选出最佳的浓缩方法为微孔滤膜吸附-洗脱法。
本文还对现有的各种肠道病毒检测方法进行了介绍比较,着重阐述了PCR检测方法的原理及其应用研究现状,在RT-PCR方法的基础上建立了水体中肠道病毒的通用引物巢式PCR检测方法,该方法主要是对RT-PCR的产物进行二次扩增,大大提高了检测的特异性和灵敏度。此外,本次研究利用巢式PCR方法对西安市各典型地表水体进行了长期定点连续监测,并对西安市水体中肠道病毒的存在情况和分布特征进行了简单分析。
At present, infection of pathogens spreaded by water is the most serious problem in the world, which has caused many epidemics, especially enteroviruses which caused epidemics more than any other viruses, and also the impacted area is the largest. So, study on the enteroviruses become a research hotspot. However, the concentration of the enteroviruses in waters are always very low, and can not be detected directly. Enteroviruses must be concentrated from water to achieve the detection level. Besides, traditional detection method of cell cuture is very complicated, and can not be used universally. Therefore, it is very useful for study on enteroviruses from waters to choose a highly effective concentration method and a sensitive detection method.
In this paper, we summarized all the concentration methods used, and we carefully chose four methods to compare,which were microporous filter adsorption-elution method, talcum powder-diatomaceous earth adsorption layer absorption-elution method and talcum powder-diatomaceous earth flocculation precipitation method. Considering about the virus recovery and reliability of the methods, we take the microporous filter adsorption-elution method for the best one. Additionally, we also summarized all the detection methods, and emphasized on the introduction of the principles of PCR and its application. Based on RT-PCR, we build the nested PCR method to detect enteroviruses in waters by universal primer, which amplificated the RT -PCR amplification, and this method could greatly increase the specificity and sensitivity. Furthermore, we deteced the typical surface water in Xi'an city consecutively by nested PCR, and made a analysis of the distrbution characteristic of enteroviruses in Xi'an city.
引文
[1]Jolis D,Robin A H,Paul A P,et al.Assessment of tertiary treatment technology for water reclamation in San Francisco,California[J].Wat.Sci.Tech,1996,33(11):181-192。
[2]WHO,Techmical Report series 639 Human Viruses in Water.1978,Water and Soil Report of a WHOScientific Group。
[3]蒋慧琴.水厂源水和出厂自来水中病毒的检测[J].病毒学杂志,1987。
[4]李荣,杨荣旺.警惕肠道病毒引起的10种感染[J].家庭教育,2005,7:30-32。
[5]张楚瑜.肠道病毒在水环境中的分布及其行为[J].生态学报,1984,(04)。
[6]肖波,赵允格,病毒在土壤和地下水中迁移研究综述[J].土壤通报,Vol.37,No.1 Feb,2006。
[7]方肇寅,宋燕燕,董虹等.北京饮用水源病毒污染研究[J].病毒学报,1992,8(8),240-244。
[8]李建标,朱美财,环境病原微生物污染与健康[J].空军总医院学报,1999,Vol(15),N0.4。
[9]李劲,李丕芬,王祖卿等.长江武汉段水中病毒污染的研究[J].环境科学学报,1987,(02)。
[10]李小锋,张楚瑜,王祖卿.武昌东湖生物污染的研究及病毒指示物的初探[J].环境科学学报,1990(04)。
[11]赵淑敏,田勇琴,邵岩,等.天津市不同水体病毒污染情况的调查[J].环境与健康杂志,1998.2。
[12]Borchardt,M.A.,Bertz,P.D.,Spencer,S.K.,Battigelli,D.A.Incidence of Enteric Viruses in Groundwater from Household Wells in Wisconsin[J].Appl.Environ.Microbiol.2003,69(2),1172-1180。
[13]M.M.Ehlers,W.O.K.Grabow,D.N.Pavlov,Dection of enteroviruses in unteated and treated drinking water supplies in South Africa[J].Water Research 39(2005)2253-2258。
[14]Eric Dubois,Ghislaine Merle,Catherine Roquier,et al.Diversity of enterovirus detected in oysters by RT-heminested PCR[J].International Journal of Food Microbiology,2004,92:35-43。
[15]仇付国,王晓昌.水环境中病毒的分布存活及其健康风险评价[J].西安建筑科技大学学报,Vol.39 No.1 Feb.2007。
[16]陈小岳,田海林.水体肠道病毒的浓集及核酸检测方法的应用[J].职业与健康,2007,23(4):293-295。
[17]J.Kellogg,et al.Immunoaffinity concentration and purification of waterborne enteric viruses for detection by RT-PCR.Appl environ Microbio1,1996,6(4):189。
[18]Fuhrman JA,Liang X,Noble RT.Rapid detection of enteroviruses in small volumes of natural waters by real-time quantitative reverse transcriptase PCR.Appl Environ Microbiol,2005,71:4523-4530。
[19]邹向,等.高效节能型饮用水净化新技术[J].水处理技术,1995,21(4):208。
[20]G.伯格(主编).环境中的病毒污染[M],第1版.北京:科学出版社,1991:11。
[21]Schwab K J,Leon R D,Sobsey M D.Concentration and purification of beef extract mock eluates from water samples for the detection of enteroviruses,hepatitis A virus,and norwalk virus by reverse transcription PCR[J].Applied and Environmental Microbiology,1995,61(2):531-537。
[22]Lewis G D,Metcalf T G.Polyethylene glycol precipitation for recovery og pathogenic viruses,including hepatitis A virus and human rotavirus,from oyster,water,and sediment samples[J].Applied and Environmental Microbiology,1988,54(8):1983-1988。
[23]Rutjes SA,Italia AR,Van den Berg HH,et al.Isolation and detection of enterovirus RNA from large-volume water samples by using the NucliSens mini MAG system and real-time nucleic acid sequence-based amplification.Appl Environ Microbiol,2005,71:3734-3740。
[24]Ma J F,Naranjo J,Gerba C P.Evaluation of MK filters for recovery of enteroviruses from tap water[J].Applied and Environmental Microbiology,1994,60(6):1974-1977。
[25]张崇淼,刘永军,王晓昌,等.环境水体中肠道病毒的膜吸附-洗脱浓缩方法的研究[J].环境科学,2007,28(7):1543-1547。
[26]郁庆福(主编).现代卫生微生物学[M].第1版.北京:人民卫生出版社,1995:62。
[27]张小英,赵祖国,王新为,等.回收污水中肠道病毒方法的改进[J].中国给水排水,2006,26(10):84-86。
[28]胡鸿雁.饮用水源水中肠道病毒的检测方法[J].环境科学与技术,2000,2(89:26-27。
[29]Lakhe S B,Paunikar W N,Parhad N M.Use of bituminous coal for concentration of enteroviruses from sewage and effluent[J].Water Research,2002,36:3298-3306。
[30]丁昌慧,邵荣标,严建国,等.细胞吸附浓集法分离外环境水中病毒[J].中国卫生检验杂志,2001,11(5):534-535。
[31]李君文,等.一种新型阳电荷滤材浓集水中F2噬菌体的效果评价.中国公共卫生,1996,12(1):26。
[32]郑耀通,林奇英,谢联辉.水体病毒浓缩条件的优化[J].中国病毒学,2004,19(1):62-66。
[33]盘宝进,刘军义,韦梅良.水体病毒浓缩方法的建立和优化研究[J].微生物学通报,2005,32(6):42-46。
[34]Bing-Mu Hsu,Chien-Hsien Chen,et al.Evaluation of enterovirus recovery in surface water by different adsorption and elution procedures[J].Chemosphere,2007,66:964-969。
[35]Khaoula Belguith,Abdennaceur Hassen,Mahjoub Aouni.Comparative study of four extraction methods for enterovirus recovery from wastewater and sewage sludge[J].Bioresource Technology,2006,97:414-419。
[36]Gilgen M,Germann D,Luthy J,et al.Three-step method for sensitive detection of enterovirus,rotavirus,hepatitis A virus,and small round structured viruses in water samples[J].Intermational Jourmal of Food Microbiology,1997,37:189-199。
[37]尤风兴,刘秉辉,张玲妹,等.水标本中脊髓灰质炎病毒三种浓缩方法的比较及改进方法的建立[J].病毒学杂志,1991,6(2):125-128。
[38]袁长青,李君文,李平.水中病毒浓集与回收的研究进展[J].中国公共卫生,1998,14(1):61-62。
[39]陈小岳,田海林.水体肠道病毒的浓集及核酸检测方法的应用[J].职业与健康,2007,23(4):293-295。
[40]Wommack K E,Colwell RR.Virioplankton:Viruses in Aquatic Ecosystems[J].Microbiol Mol Biol Rev,2000,64(1):69。
[41]李建标,朱美财,环境病原微生物污染与健康[J].空军总医院学报,1999,,Vol(15),No.4。
[42]宋农,等.金葡萄固相抗体吸附水样中F2噬菌体的效果[J].中国公共卫生学报,1997,16(1):40。
[43]Espinoza J C,Kuznar J.Rapid simultaneous detedtion and quantitation of infectious pancreatic necrosis virus[J].Journal of Virological Methods,2002,105:81-85。
[44]马骢,马学斌.海水中病毒的检测与分布研究进展[J].解放军预防医学杂志,2007,25(2):145-148。
[45]Venter J C Remington K,Heidellberg JF,et al.Environmental genome shotgun sequencing of the Sargasso Sea[J].Science,2004,304(5667):66。
[46]周德庆编.微生物学教程[M],第四版.北京:高等教育出版社,2005。
[47]岳春梅,明镇寰.分子生物学技术在环境微生物监测中的应用[J].微生学通报,2000,27(2):215-218。
[48]柯昌文.人肠道病毒分类和鉴别方法研究进展[J].中国计划免疫,2006,12(6):515-518。
[49]Oberste MS,Maher K,Flemister MR,et al.Comparison of classic and molecular approaches for the identification of umtypable enteroviruses[J].J Clin Microbiol,2000,38:1170-1174。
[50]Oberste MS,Maher K,Flemister MR,et al.Improved molecular identification of enteroviruses by RT-PCR and amplicon sequencing[J].J Clin Virol,2003,26:375-377。
[51]Taylor M B,Cox N,Very M A,et al.The occurrence of hepatitis A and astroviruses in selected iver and dam waters in South Africa[J].Water Research,2001,35:2653-2660。
[52]费选文,林祥伟,谢若男,等.应用4种方法检测肠道病毒的结果分析[J].中华实验与临床病毒学杂志,2003,1:45-46。
[53]Formiga CM,Hundesa A,Clemente C P,et al.Nested multiplex PCR assay for detection of human enteric viruses in shellfish and sewage[J].Virol Methods,2005,125:111-118。
[54]Guyader F,Dubios E,Menard D,et al.Detection of hepatitis A virus,rotavirus,and enterovirus in maturally contaminated shellfish and sediment by reverse transcription-seminested PCR[J].Appl Environ Microbiol,1994,60:3665-3671。
[55]牛玉宏,杨英珍,虞勇,等.肠道病毒基因实时定量RT-PCR方法的建立.复旦学报(医学科学版),2001,28(6):542-544。
[56]郑武飞主编.医学免疫学.北京:人民卫生出版社,1989.
[57]马军,邱立平,冯琦.聚合酶链式反应在微生物检测中的应用[J].中国给水排水,2002,18(1):34-36。
[58]Zehr J P,Mc Reynolds L A.Use of degenerate oligonucleotides for amplification of the nif H gene from the marine cyanobacterium trichodesmium thiebautii[J].Appl Environ Microbiol,1989,55(10):2522-2526。
[59]Catalan V,Garcia F,Moreno C,et al.Detection of Legionella pneumopHila in wastewater by nested polymerase chain reaction[J].Research im Microbiology,1997,148(1):71-78。
[60]Catalan V,Garcia F,Dasi M A,et al.Nested polymerase chain reaction for detection of 0Legionella pneumopHila in water[J].Research im Microbiology,1994,145(3):603-610。
[61]Fricker E J,Munm K,Fricker C R.The use of PCR for the detection of bacteria and viruses [J].Water Supply,2000,17(2):5-16。
[62]Heiber I,Frahm E,Obst U.Molecular biological detection of hygienically relevant microorgamisms in water samples based on specific nucleic acid probes and magnetic beads [J].Water Supply,1999,17(2):1-3。
[63]袁长青,叶建锋,李君文.改进的PCR技术快速检测水中肠道病毒的研究[J].中国卫生检验杂志,2008,10(4):395-399。
[64]程莉,何晓青,李伟,等.北方某城区水环境中轮状病毒的初步检测[J].生态毒理学报,2007,2(2):202-206。
[65]张崇淼,刘永军,王晓昌,等.逆转录聚合酶技术同时检测水中多种肠道病毒[J].环境科学研究,2007,20(3):137-141。
[66]刘永军,张崇淼,王晓昌,等.通用引物PCR方法在地表水病原菌检测中的应用[J].环境科学研究,2007,20(2):89-94。
[67]王稳,薄志坚,吕作芝,等.应用逆转录PCR检测海产贝壳类中的甲型肝炎病毒[J].微生物学免疫学进展,2006,34(2):19-23。
[68]寇晓霞,吴清平,范宏英,等.水体中诺沃克病毒RT-PCR检测研究[J].微生物通报,2007,34(4):650-653。
[69]裘湛,闻岳,黄翔峰等.PCR-DGGE技术在水解酸化缺氧法处理采油废水的微生物研究中的应用[J].环境污染与防治,2006,28(6):439-442。
[70]王峰,傅以钢,夏四清,等.PCR-DGGE技术在城市污水化学生物絮凝处理中的特点[J].环境科学,2004,4。
[71]袁宏林,刘永军,王晓昌等.生物造粒流化床污水处理反应器的微生物多样性[J].生态学杂志,2007,26(8):1199-1203。
[72]陆德源主编.医学微生物学,第四版.北京:人民卫生出版社,1996。
[73]蔡霞.定量PCR技术及其应用现状.现代诊断与治疗[J].2005,16(2)。
[74]吴小兵,董小岩,伍志坚,等.一种快速高效分离腺病毒伴随病毒的方法[J].科学通报,2000,45(19):2071-2075。
[75]彭建强,等.一种高效快速浓缩腺相关病毒载体的方法[J].病毒学报,2005,21(1):9-14。
[76]SPSS12.0 For Windows实用教程与操作技巧[M].第1版.上海科学技术文献出版社,2005。
