| 202 | 2 | 41 |
| 下载次数 | 被引频次 | 阅读次数 |
为了建立特异、灵敏、快速的荧光定量RT-PCR方法用于检测埃可病毒9型病毒核酸,并初步应用于埃可病毒9型的临床标本检测。根据GenBank登录的埃可病毒9型毒株VP1基因序列,应用生物学软件进行序列比对,在保守区设计特异性引物和TaqMan探针。对反应条件进行优化,验证方法的特异性、灵敏度和重复性,同时对疑似埃可病毒9型病例标本进行检测。该方法对埃可病毒9型的检出有高度特异性,与EV71、CA16、CA24v、CA6、CA10、埃可病毒30型等均无交叉反应,检测灵敏度均达0.1TCID50/mL,可从疑似埃可病例粪便标本中直接检测病毒核酸,检测仅需3~4h。本研究建立的埃可病毒9型TaqMan荧光定量RT-PCR检测方法特异、灵敏,适用于临床早期诊断。
Abstract：To establish a TaqManTM-based real-time reverse transcription-polymerase chain reaction(RTPCR)for detection of the Echovirus 9 serotype.The established method was used primarily to test clinical samples for Echovirus 9.The gene sequences of Echovirus 9 were downloaded from GenBank.They were aligned using biologic software,and specific primers and probes were designed in the conserved region of the VP1 gene for the Echovirus 9.The primers,probes and reaction conditions were optimized to improve the sensitivity and specificity of real-time RT-PCR.Clinical specimens were collected from an epidemic of viral encephalitis in children underwent real-time RT-PCR.The specificity of the RT-PCR was high,with no cross reactions with EV71,CA16,CA24 v,CA6,CA10,ECHO-6 or ECHO-30.The sensitivity of realtime RT-PCR was 0.1 TCID50/mL,and viral RNA could be detected directly from clinical specimens.It took 34 hours to complete real-time RT-PCR.This TaqManTM-based real-time RT-PCR was a rapid,sensitive and specific method for the molecular diagnosis of the Echovirus 9 serotype.
[1]Akasu Y.Outbreak of aseptic meningitis due to ECHO-9in northern Kyushu island in the summer of 1997[J].Kurume Med J,1999,46(2):97-104.
[2]Centers for Disease Control and Prevention(CDC).Outbreaks of aseptic meningitis associated with echoviruses9and 30and preliminary surveillance reports on enterovirus activity,United States,2003[J].Morb Mortal Wkly Rep,2003,52(32):761-764.
[3]Figueiredo C A,Luchs A,Russo D H,de Cassia Compagnoli Carmona R,Afonso A S,de Oliveira M I,Curti S P,de Moraes J C,Toscano C M,Ciccone F H,Timenetsky M T.Rubella virus genotype 1Gand echovirus 9as etiologic agents of exanthematous diseases in Brazil:insights from phylogenetic analysis[J]Arch Virol,2014,159(6):1445-1451.
[4]Minnaar R P,Koen G,de Haan K,Wolthers K C,Benschop K M.Evaluation of two(semi-)nested VP1based-PCRs for typing enteroviruses directly from cerebral spinal fluid samples[J].J Virol Methods,2012,185(2):228–233.
[5]Tryfonos C,Richter J,Koptides D,Yiangou M,Christodoulou C G.Molecular typing and epidemiology of enteroviruses in Cyprus,2003–2007[J]J Med Microbiol,2011,60(10):1433-1440.
[6]朱冰,钟家禹,夏慧敏,龚四堂,肖密丝,谢嘉慧,张莹莹,华亮,连广琬.2008年广州地区手足口病的病原学研究[J].中华儿科杂志,2010,48(2):127-130.
[7]马芳,李明星,李丽,张征,杨炬,杜建才.宁夏一起埃可病毒9型病毒性脑膜炎暴发疫情的流行病学分析[J].宁夏医科大学学报,2011,33(1):44-47.
[8]汪照国,刘晓琳,杨婷婷,弋英.2008-2009年青岛地区手足口病病原学的调查分析[J].病毒学报,2011,27(5):438-441.
[9]邱晓枫,谢立,黄志成,杨旭辉,王婧,潘劲草.某幼儿园一起脑炎疫情流行病学及病原研究[J].中国卫生检验杂志,2014,24(3):309-315.
[10]许云霞,丁爱仙.一起埃可病毒9型所致感染性疾病的暴发调查[J].江苏预防医学,2016,27(1):95-96.
[11]Shaukat S,Angez M,Alam M,Jebbink M,Deijs M,Canuti M,Sharif S,de Vries M,Khurshid A,Mahmood T,van der Hoek L,Zaidi S.Identification and characterization of unrecognized viruses in stool samples of non-polio acute flaccid paralysis children by simplified VIDISCA[J].Virol J,2014,11(1):146.
[12]Fujimoto,T,Izumi,H,Okabe,N,Enomoto,M,Konagaya,M,Chikahira,M,Munemura,T,Taniguchi,K.Usefulness of real-time reverse transcriptionpolymerase chain reaction for the diagnosis of echovirus aseptic meningitis using cerebrospinal fluid[J].Jpn J Infect Dis,2009,62(6)455-457.
[13]World Health Organization.Manual for the virological investigation of poliomyelitis[M].Geneva:World Health Organization,2000:78-96.
[14]Tanaka T,Takada T,Takagi D,Takeyama N,Kitazawa Y.Acute renal failure due to rhabdomyolysis associated with echovirus 9infection:a case report and review of literature[J].Jpn J Med,1989,28(2):237-242.
[15]Zuckerman M,Sheaff M,Martin J,Gabriel C.Fatal case of echovirus type 9encephalitis[J].J Clin Pathol,1993,46(9):865-866.
[16]Oberste M,Maher K,Kilpatrick D,Pallansch M.Moleculare volution of the human entero viruses:correlation of serotype with VPl sequence and application to picornavirus classification[J].J Virol,1999,73(3):1941-1948.
基本信息:
DOI：10.13242/j.cnki.bingduxuebao.003240
中图分类号:R440;R512.5
引用信息:
[1]邱晓枫,张国忠,黄志成,等.埃可病毒9型荧光RT-PCR快速检测方法的建立及应用[J].病毒学报,2017,33(06):836-841.DOI:10.13242/j.cnki.bingduxuebao.003240.
基金信息:
杭州市科技发展计划项目(项目号:20120633B22);题目:埃可病毒9型的快速检测方法建立以及流行特征研究; 杭州市农业与社会发展科研计划项目(项目号:20170533B76);题目:杭州市区手足口病患儿中埃可病毒的检测及其分子特征研究~~
2017-10-19
2017-10-19
2017-10-19