杨丽;何晓青;韦玉梅;朱轶;程莉;王子健;黄文;

• 1:北京林业大学生物科学与技术学院
• 2:中国科学院生态环境研究中心环境水质学国家重点实验室
• 3:华中农业大学食品科技学院

摘要(Abstract)：

为快速高效地检测人星状病毒,建立了一种新的试验方法——环介导等温扩增技术(loop-mediated isothermal amplification technology,LAMP),并对人星状病毒LAMP反应条件进行了优化。结果表明,镁离子终浓度为4mmol·L-1、甜菜碱终浓度为1mol·L-1的25μL体系下65℃反应90min为其最佳反应条件。对扩增终产物进一步进行酶切分析得到的条带大小与预期的结果(84和135bp)吻合,以人星状病毒、轮状病毒及诺如病毒为模板进行特异性测试亦没有发生交叉反应。将人星状病毒质粒等倍稀释后分别用LAMP及聚合酶链式反应(PCR)检测其灵敏度,结果表明,LAMP与PCR的灵敏度分别达到5.04copies·μL-1和50.4copies·μL-1。用改良的逆转录环介导等温扩增技术(RT-LAMP)及逆转录PCR(RT-PCR)检测12个再生水水样,其中人星状病毒检出率为分别为41.7%(5/12)和33.3%(4/12)。以上结果证明,LAMP是一种检测程序简单、灵敏度和特异性较高的检测手段,在人星状病毒的快速检测方面具有一定的开发潜力。

关键词(KeyWords)： 人星状病毒;环介导等温扩增技术(LAMP);HNB;焦磷酸镁;再生水

Abstract:

Keywords:

基金项目(Foundation): 国家水体污染控制与治理科技重大专项(2009ZX07527-005);; 国家"863"重点项目(2008AA062501);; 中国科学院生态环境研究中心环境水质学国家重点实验室开放基金课题(2009-001)

作者(Author): 杨丽;何晓青;韦玉梅;朱轶;程莉;王子健;黄文;

Email:

参考文献(References)：

• [1]Rungnapa M,Niwat M,Pattara K,et al.2008.Genetic diver-sity of norovirus,sapovirus,and astrovirus isolated from chil-dren hospitalized with acute gastroenteritis in Chiang Mai,Thailand[J].Journal of Medical Virology,80(10):1749-1755
• [2]Tuan A N,Le P H,Le D P,et al.2008.Identification of human astrovirus infections among children with acute gastro-enteritis in the southern part of Vietnam during2005-2006[J].Journal of Medical Virology,80(2):298-305
• [3]Santos R A,Borges A M,Da Costa P S,et al.2007.Astrovirus infection in childroen living in the central west region of Brazil[J].Memórias do Instituto Oswaldo Cruz,102(2):209-213
• [4]WHO.2010.Guidelines for Drinking-Water Quality[M].World Health Organization,171-207
• [5]Pusch D,Oh D Y,Wolf S,et al.2005.Detection of enteric viruses and bacteria indicators in German environmental wa-ters[J].Archives of Virology,150(5):929-947
• [6]何晓青.2008.三种典型肠病毒的分子检测及其在北京地区中的来源与分布规律[D].北京:中国科学院生态环境研究中心,32-92He X Q.2008.Molecular Detection of Three Enteric Viruses and Their sources and Distribution in Environmental Water Samples of Beijing Region[D].Beijing:Research Center for Eco-Environmental Sciences,Chinese Academy of Sci-ences.32-92(in Chinese)
• [7]程莉.2010.北京地区水环境中病原细菌及典型肠病毒污染特征的研究[D].北京:北京林业大学,26-88Cheng L.2010.Pollution Characteristics of Pathogenic Bacteria and Three Enteric Viruses in Environmental Water Samples of Beijing[D].Beijing:Beijing Forestry University,26-88(in Chinese)
• [8]Notomi T.2000.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Research,28(12):63-63
• [9]Nagamine K,Hase T,Notomi T.2002.Accelerated reaction by loop-mediated isothermal amplification using loop primers[J].Molecular and Cellular Probes,16(3):223-229
• [10]Nagamine K,Watanabe K,Ohtsuka K,et al.2001.Loop-me-diated isothermal amplification reaction using a nondenatured template[J].Clinical Chemistry,47(9):1742-1743
• [11]王丽,石磊,李琳.2006.DNA环介导的恒温扩增法在快速鉴定病原微生物中的应用[J].生命化学,26(5):462-465
• [12]Mori Y,Kitao M,Tomita N,et al.2004.Real-time turbidime-try of LAMP reaction for quantifying template DNA[J].Jour-nal of Biochemical and Biophysical Methods,59(2):145-157
• [13]Motoki G,Eiichi H,Atsuo O,et al.2009.Colorimetric detec-tion of loop-mediated isothermal amplification reaction by using hydroxyl naphthol blue[J].BioTechniques,46(3):167-172
• [14]聂凯,王大燕,秦萌,等.2010.基于颜色判定的环介导逆转录等温扩增技术检测人甲型H1N1流感病毒基因[J].病毒学报,26(2):81-87Nie K,Wang D Y,Qin M,et al.2010.Colorimetric detec-tion of human influenza A H1N1virus by reverse transcrip-tion loop mediated isothermal amplification[J].Chinese Journal of Virology,26(2):81-87(in Chinese)
• [15]秦成峰,秦鄂德.2007.环介导的恒温扩增技术及其在病毒性疾病诊断中的应用[J].生物技术通讯,18(5):859-862Qin C F,Qin E D.2007.Loop-mediated isothermal am-plification technology and its application in virus detec-tion[J].Letters in Biotechnology,18(5):859-862
• [16]李启明,马学军,周蕊,等.2006.环介导逆转录等温扩增技术(RT-LAMP)在丙型肝炎病毒基因检测中的应用[J].病毒学报,22(5):334-338Li Q M,Ma X J,Zhou R,et al.2006.Detection of HCV gene by reverse transcription loop-mediated isothermal amplification method[J].Chinese Journal of Virology,22(5):334-338(in Chinese)
• [17]Katayama H,Shimasaki A,Ohgaki S.2002.Development of a virus concentration method and its application to detection of enterovirus and norwalk virus from coastal seawater[J].Applied Environmental Microbiology,68(3):1033-1039
• [18]Hill J,Beriwal S,Chandra I,et al.2008.Loop-mediated iso-thermal amplification assay for rapid detection of common strains of Escherichia coli[J].Journal of Clinical Microbiol-ogy,46(8):2800-2804
• [19]Curtis K A,Rudolph D L,Owen S M.2008.Rapid detection of HIV21by reverse transcription loop-mediated isothermal amplification(RT-LAMP)[J].Journal Virological Meth-ods,51(2):264-270
• [20]吴绍强,孙晓智,林祥梅,等.2008.亚洲Ⅰ型口蹄疫病毒环介导等温扩增(LAMP)检测方法的建立[J].检验检疫科学,18(1):9-12Wu S Q,Sun X Z,Lin X M,et al.2008.Loop-mediated iso-thermal amplification of foot-and-mouth disease virus sero-type Asia I[J].Inspection and Quarantine Science,18(1):9-12(in Chinese)
• [21]Karra S,Katsivela E.2007.Microorganisms in bioaerosol emis-sions from wastewater treatment plants during summer at a Mediterranean site[J].Water Research,41(6):1355-1365
• [22]吕欣,尹文,仇付国,等.2004.几种水处理方法对污水中病毒清除作用的评估[J].中国公共卫生,20(11):1281-1282Lv X,Yi W,Qiu F G,et al.2004.Evaluation on inacti-vation effectiveness of different water treatments on en-teric viruses in wastewater[J].Chinese Journal of Pub-lic Health,20(11):1281-1282(in Chinese)