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双重荧光定量RT-PCR法检测柯萨奇病毒A2和A5型 预览 被引量:3

Double fluorescent quantitative RT-PCR assay for the detection of coxsackievirus A2 and A5 subtypes
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摘要 目的建立同时检测柯萨奇病毒A2和A5型(CVA2、CVA5)的双重实时荧光定量RT-PCR法。方法用Primer Express3.0软件设计高度特异性引物和荧光标记探针。通过优化反应体系,建立标准曲线,并评价双重荧光定量RT-PCR法的特异性、灵敏度和重复性。用建立的方法检测367例疑似手足口病患儿的粪便标本,并对阳性结果测序验证。结果荧光定量RT-PCR结果表明本实验所选的引物和探针可特异性检测并区分出CVA2、CVA5亚型,与其他肠道病毒的阳性核酸未发生交叉反应。该方法最低检测限达到10^2copies/m L,批内变异系数(CV)均〈1.49%。367份临床标本中CVA2型阳性23份,阳性率6.3%;CVA5阳性11份,阳性率3%,检测阳性结果与测序结果一致。结论成功建立了在单管中同时检测并鉴别CVA2、CVA5型的双重实时荧光定量RT-PCR检测方法。该方法具有较好的灵敏度、特异性、重复性,可用于CVA2、CVA5型引起的暴发疫情的快速筛查和手足口病的流行病学的研究。 Objective To establish a double fluorescent quantitative RT-PCR assay for simultaneously detecting coxsackievirus A2( CVA2) and A5( CVA5) subtypes. Methods The specific primers and probes of CVA2 and CVA5 subtypes for fluorescent quantitative RT-PCR assay were designed with Primer Express 3. 0 software. The optimizing double fluorescent quantitative RT-PCR system with a standard curve was established,and its specificity,sensitivity and repeatability were evaluated. Then,the stool specimens from367 children suspected with hand-foot-and-mouth disease were detected by this assay,and the positive specimens were further sequenced. Results The established double fluorescent quantitative RT-PCR was able to detect CVA2 and CVA5 subtypes specifically,and had no cross reaction with other enteric viruses. Its lowest detectable limit was 102 copies / m L,and the intra assay coefficients of variation were all less than 1. 49%. The positive rates of CVA2 and CVA5 subtypes in 367 clinical specimens were 6. 3%( 23 /367)and 3%( 11 /367),respectively,and the positive results were verified by subsequent sequencing. Conclusion The double fluorescent quantitative RT-PCR assay for simultaneously detecting CVA2 and CVA5 subtypes in a single test tube is successfully established,which has good specificity,sensitivity and repeatability,and may be used to rapidly screen the infections caused by CVA2 and CVA5 subtypes and investigate the epidemiology of hand-foot-and-mouth disease.
作者 李静云 崔大伟 谢国良 成军 孙长贵 王国政 金美彤 沈雄文 杨先知 陈瑜 LI Jing-yun, CUI Da-wei, XIE Guo-liang, CHENG Jan, SUN Chang-gui, WANG Guo-zheng, JIN Mei-tong, SHEN Xiong-wen, YANG Xian-zhi , CHEN Yu ( 1. Department of Clinical Laboratory, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang ; 2. Clinical Experimental Center, ll7th Hospital of PLA, Hangzhou 310013, Zhejiang , China)
出处 《临床检验杂志》 CAS CSCD 2015年第5期321-324,共4页 Chinese Journal of Clinical Laboratory Science
基金 “十二五”国家科技重大专项(2012ZX10004-210)
关键词 手足口病 荧光定量PCR 柯萨奇病毒 柯萨奇病毒A2型 柯萨奇病毒A5型 hand-foot-and-mouth disease fluorescent quantitative RT-PCR coxsackievirus coxsackievirus A2 subtype coxsackie virus A5 subtype
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参考文献9

  • 1Ang I ,W, Koh HK, Cluui KP, et al, Epidenuology and control ofhand, foot and mouth (iisease in Singapore, 2001-2007 [J]- AnnAcad Med Singapore, 2009 , 38(2):106-112. 被引量:1
  • 2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(2):127-130. 被引量:38
  • 3Vuorinen T, Osterhack K, Kuisma J, et al. Epididymitis caused bycoxsackievirus A6 in association with hand, foot, and mouth disease[J]. J Clin Microi)iol, 2014, 52(12):44124413. 被引量:1
  • 4Hlomqvist S, Klemola P, Kaijalainen S, et al. Co-circulation of cox-sackieviruses A6 and A10 in hand, foot and mouth disease outbreakin Finland : J]. J Clin Virol, 2010,48( 1 ) :49-54. 被引量:1
  • 5YipCC, Uu SK, o. PC, et al. Recombinant coxsackievirus A2and deaths of children, I long Kong, 2012 [ J]. .2merg Infect Dis,2013,19(8):1285-1288. 被引量:1
  • 6Du J , Wang X,Hu Y,et nl. (Changing aetiology of hand , for>{ andmouth disease in Linyi, China, 2009-2011 [ J ] . Clin Microbiol In-fect, 2014, 20(1):4749. 被引量:1
  • 7Park SH , Choi SS, Oh SA , et al. Detection and characterization ofenterovirus associated with herpangina and hand, foot, and mouthdisease in Seoul, Korea [ J]. Clin Lab, 2011 , 57 ( 11-12) :959-967. 被引量:1
  • 8Park K, I^ee B, Baek K, et al. Enteroviruses isolated from herpangi-na and hand-foot-and-mouth disease in Korean children [J]. Virol J ,2012, 9:205. 被引量:1
  • 9Puenpa J, Mauleekoonphairoj J, Linsuwanan P, et al. Prevalenceand characterization of enterovinjs infections among pediatric patientswith hand fool mouth disease, herpangina and influenza like illness inThailand, 2012 [J]. l)LoS One, 2014, 9(6) :e98888. 被引量:1

二级参考文献8

  • 1AbuBakar S, Chee HY, Al-Kobaisi MF, et al. Identification of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalomyelitis in Malaysia. Virus Res, 1999,61(1) :1-9. 被引量:1
  • 2Lin TY, Chang LY, Hsia SH, et al. The 1998 enterovins 71 outbreak in Taiwan: pathogenesis and management Clin Infect Dis, 2002,34 (Suppl 2) :S.52-57. 被引量:1
  • 3Yan JJ, Su U, Chen PF, et al. Complete genome analysis of enterovirus 71 isolated from an outbreak in Taiwan and rapid identification of enterovims 71 and coxsackievirus A16 by RT-PCR. J Med Virol,2001,65 :331-339. 被引量:1
  • 4Ahmad K. Hand, foot, and mouth disease outbreak reported in Singapore. Lancet, 2000, 356 : 1338. 被引量:1
  • 5Li L, He Y, Yang H, et al. Genetic characteristics of human enterovirus 71 and coxsackievirus A16 circulating from 1999 to 2004 in Shenzhen, People's Republic of China. J Clin Microbiol, 2005,43:3835-3839. 被引量:1
  • 6Mackay IM, Arden KE, Nitsche A. Real-time PCR in virology. Nucleic Acids Res, 2002,30:1292-1305. 被引量:1
  • 7Petitjean J, Vabret A, Dina J, et al. Development and evaluation of a teal-time RT-PCR assay on the LightCyeler for the rapid detection of enterovirus in cerebrospinal fluid specimens. J Clin Virol, 2006,35:278-284. 被引量:1
  • 8Tan EL, Yong LL, Quak SH,et al. Rapid detection of enterovirus 71 by real-time TaqMan RT-PCR. J Clin Virol, 2008,42:203- 206. 被引量:1

共引文献37

同被引文献24

  • 1Thao NT, Ngoc NT, Tu PV, et al. Development of a multiplex poly- merase chain reaction assay for simultaneous identification of human enterovirus 71 and coxsackievirus A16 [ J]. J Virol Methods, 2010, 170(1-2) : 134-139. 被引量:1
  • 2He YQ, Chen L, Xu WB, et al. Emergence, circulation and spatio- temporal phylogenetic analysis of Coxsackievirus A6 and Coxsackievir- us A10 associated hand, foot and mouth disease infections from 2008 to 2012 in Shenzhen, China[J]. J Clin Microbiol, 2013, 51 (11): 3560-3566. 被引量:1
  • 3Oberste MS, Maher K, Williams A J, et al. Species-specific RT-PCR amplification of human enteroviruses : a tool for rapid species identifi- cation of uncharacterized enteroviruses [J]. J Gen Virol, 2006, 87 (Pt 1) : 119-128. 被引量:1
  • 4Kobayashi M, Makino T, Hanaoka N, et al. Clinical manifestations of coxsackievirus A6 infection associated with a major outbreak of hand, foot, and mouth disease in Japan[J]. Jpn J Infect Dis, 2013, 66(3) : 260-261. 被引量:1
  • 5Blomqvist S, Klemola P, KaijaIainen S, et al. Co-circulation of cox- sackieviruses A6 and A10 in hand, foot and mouth disease outbreak in Finland[J]. J Clin Virol, 2010, 48(1) : 49-54. 被引量:1
  • 6Puenpa J, Chieochansin T, Linsuwanon P, et al. Hand, foot, and mouth disease caused by coxsackievirus A6, Thailand, 2012 [ J]. Emerg Infect Dis, 2013, 19(4) : 641-643. 被引量:1
  • 7Montes M, Artieda J, Pineiro L D, et al. Hand, foot, and mouth disease outbreak and coxsackievirus A6, northern Spain, 2011 [J]. Emerg Infect Dis, 2013, 19(4) : 676-678. 被引量:1
  • 8Mirand A, Henquell C, Archimbaud C, et al. Outbreak of hand, foot and mouth disease/herpangina associated with coxsackievirus A6 and A10 infections in 2010, France: a large citywide, prospective observational study [ J ]. Clin Microbiol Infect, 2012, 18 ( 5 ) : EllO-Ell8. 被引量:1
  • 9Lu QB, Zhang XA, Wo Y, et al. Circulation of Coxsackievirus AIO and A6 in hand-foot-mouth disease in China, 2009-2011 [ J]. PLoS One, 2012, 7(12) : e52073. 被引量:1
  • 10李玉运,朱汝南,钱渊,邓洁,孙宇,王芳,赵林清,刘立颖.肠道病毒71型北京分离株VP4基因序列及蛋白抗原性分析[J].病毒学报,2011,27(3):207-214. 被引量:5

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