| 326 | 5 | 46 |
| 下载次数 | 被引频次 | 阅读次数 |
罗氏沼虾白尾病(White tail disease,WTD)是流行于罗氏沼虾苗种阶段的重要疾病,该病病原被确定为罗氏沼虾野田村病毒(Macrobrachium rosenbergii Nodavirus,MrNV),为了建立罗氏沼虾野田村病毒中国分离株(MrNV-chin)的便捷、灵敏、特异的分子诊断方法,本研究根据罗氏沼虾野田村病毒RNA2基因保守序列的6个区域设计了4条特异性引物和1条检测探针,将环介导等温扩增技术与侧向流层析试纸(Lateral flow dipstick,LFD)相结合,建立了MrNV的RT-LAMP-LFD检测方法。结果表明,该方法的灵敏度是常规琼脂糖凝胶电泳的10倍;从核酸提取到检测结果判定仅需45min;反应特异性强,对其他虾类病毒WSSV、IHHNV、IMNV、TSV和YHV扩增结果均为阴性;操作简单,不需要复杂的仪器,在61℃水浴反应即可;利用LFD试纸显示结果,肉眼可直接观察判定。作为一种快速灵敏实用的分子诊断技术,该方法有利于罗氏沼虾白尾病的诊断与预防。
Abstract：White coloration of the muscle of the giant river prawn(Macrobrachium rosenbergii)is a serious problem in China.The Macrobrachium rosenbergii Nodavirus(MrNV)has been confirmed to be the pathogen that causes this disorder.To develop a rapid,sensitive and specific technology for the detection of Macrobrachium rosenbergii Nodavirus isolated from China(MrNV-China),a reverse-transcription loopmediated isothermal amplification assay combined with a lateral flow dipstick(RT-LAMP-LFD)assay method is described.A set of four primers and a labeled probe were designed specifically to recognize six distinct regions of the MrNV RNA2 gene.Results showed the sensitivity of the RT-LAMP-LFD assay was ten-times higher than the reverse-transcription loop-mediated isothermal amplification assay(RT-LAMP)with agarose gel electrophoresis.The assay was conducted with one-step amplification at 61℃in a single tube within 45 min.No product was generated from shrimps infected with other viruses,including DNA viruses(infectious hypodermal and hematopoietic necrosis virus(IHHNV);white spot syndrome virus(WSSV))and RNA viruses(Taura syndrome virus(TSV);infectious myonecrosis virus(IMNV);yellow head virus(YHV)).Results were visualized by the LFD method.Therefore,the described rapid and sensitive assay is potentially useful for MrNV detection.
[1]Sahul Hameed A S,Yoganandhan K,Sri Widada J,et al.Experimental transmission and tissue tropism of acrobrachium rosenbergii nodavirus(MrNV)and its associated extra small virus(XSV)[J].Dis Aquat,2004a,62:191-196.
[2]石正丽,解云礼,汤显春,等.罗氏沼虾诺达病毒的核酸检测及其部分序列分析[J].病毒学报,2004,20(1):62-66.
[3]Sri Widada J,Richard W,Cambournac I,et al.Genome based detection methods of Macrobrachium rosenbergii nodavirus,apathogen of the giant freshwater prawn,Macrobrachium rosenbergii dot-blot,in situ hybridization and RT-PCR[J].J Fish Dis,2003,26(10):583-590.
[4]Sri Widada J,Richard V,Shi Z L,et al.Dot-blot hybridization and RT-PCR detection of extra small virus(XSV)associated with white tail disease of prawn Macrobrachium rosenbergii[J].Dis Aquat Organ,2004,58(1):83-87.
[5]Romestand B,Bonami J R.A sandwich enzyme linked immunosorbent assay(S-ELISA)for detection of MrNV in the giant freshwater prawn,Macrobrachium rosenbergii(de Man)[J].J Fish Dis,2003,26(2):71-75.
[6]NOTOMI T.Loop-mediated isothermal amplification[J].Nippon Rinsho,2007,65(5):957-961.
[7]Guatelli J C,Whitfield K M,kwoh D Y,et al.Isothermal,in vitro amplification of nucleic acids by multienzyme reaction modeled after retroviral replication[J].Proc Natl Acad Sci,1990,87:1874-1878.
[8]Cheng W,Chen J C.Isolation and characterization of an Enterococcus-like bacterium causing muscle necrosis and mortality in Macrobrachium rosenbergii in Taiwan[J].Dis Aquat Organ,1998,34(2):93-101.
[9]Jayaprakash N S,Pai S S,Philip R,et al.Isolation of a pathogenic strain of Vibrio alginolyticus from necrotic larvae of Macrobrachium rosenbergii(de Man)[J].J Fish Dis,2006,29(3):187-191.
[10]Puthawibool T,Senapin S,Flegel T W,et al.Rapid and sensitive detection of Macrobrachium rosenbergii nodavirus in giant freshwater prawns by reverse transcription loop-mediated isothermal ampli fication combined with a lateral flow dipstick[J].Mol Cell Probes,2010,24:244-249.
[11]Qian D,Shi Z,Zhang S,et al.Extra small virus-like particles(XSV)and nodavirus associated with whitish muscle disease in the giant freshwater prawn,Macrobrachium rosenbergii[J].J Fish Dis,2003,26(9):521-527.
[12]Hsu J P,Liu C I.Studies on yeast infection in cultured giant freshwater prawn(Macrobrachium rosenbergii)[J].Fish Dis Res,1994,15:55-68.
[13]Lacroix D,Glude J,Thomas J E,et al.From research to commercialization:lessons from four different strategies in the development of freshwater prawn culture(Macrobrachium rosenbergii)in America since 1977[J].World Aquaculture,1994,25(1):5-17.
[14]Savan R,Igarashi A,Matsuoka S,et al.Sensitive and rapid detection of Edwardsiellosis in fish by a loop-mediated isothermal amplification method[J].Appl Environ Microbiol,2004,70(1):621-624.
[15]Kiatpathomchai W,Jaroenram W,Arunrut N,et al.Shrimp Taura syndrome virus detection by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick[J].J Virol Methods,2008,153(2):214-217.
[16]Jaroenram W,Kiatpathomchai W,Flegel T W.Rapid and sensitive detection of white spot syndrome virus by loop-mediated isothermal amplification combined with a lateral flow dipstick[J].Mol Cell Probe,2009,23(2):65-70.
[17]Puthawibool T,Senapin S,Kiatpathomchai W,et al.Detection of shrimp infectious myonecrosis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick[J].J Virol Methods,2009,156(1/2):27-31.
基本信息:
DOI：10.13242/j.cnki.bingduxuebao.002539
中图分类号:S945.41
引用信息:
[1]林锋,刘莉,郝贵杰,等.罗氏沼虾野田村病毒中国株RT-LAMP-LFD快速检测方法的研究[J].病毒学报,2014,30(05):502-507.DOI:10.13242/j.cnki.bingduxuebao.002539.
基金信息:
农业部公益性行业专项(201103034); 湖州市科技攻关项目(2012GN08); 浙江省淡水养殖科技创新团队项目(2012R10026-11)
2014-09-23
2014-09-23
2014-09-23