期刊文献+

荧光定量RT-PCR在轮状病毒检测中的应用 被引量:3

Quantitation of rotavirus by real time reverse transcription polymerase chain reaction
下载PDF
导出
摘要 目的:建立轮状病毒的荧光定量RT-PCR检测方法,用于检测临床腹泻样本。方法:使用Taqman探针技术,建立轮状病毒VP6特异的荧光定量RT-PCR体系。与WHO推荐的轮状病毒检测方法ELISA平行检测,对该体系的检测范围、灵敏度、特异性等参数进行评价。并将该体系用于临床腹泻样本。结果:该荧光定量RT-PCR体系具有高度灵敏度和特异性,线性范围宽,最低可检出1TCID50/ml。对113份临床腹泻样本的平行检测结果显示,该荧光定量RT-PCR体系与ELISA的检出率差异无统计学意义(χ2=0.41,P>0.5),可同样用于临床样本检测。结论:成功建立了轮状病毒荧光定量RT-PCR体系,该体系灵敏、特异、重复性好,可用于临床检测。 Objective: To develop a more sensitive and specific diagnostic test for rotavims by applying real time quantitative RT-PCR technology. Methods: The real time RT-PCR specific for VP6 gene of rotavirus was developed by applying Taqman technology. And the parameters of the developed real time RT-PCR, including sensitivity, specificity and reproducibility were evaluated. Meanwhile, ELISA was used as a parallel detection method to detect RV in stock and clinical fecal specimen since it was recommended as a standard method for RV detection by WHO. Results: The detection limit of the real time quantitative RT-PCR was 1TCID50/ml, which was llog more sensitive than ELISA. The relationship between threshold cycle (Ct) and amount of virus was linear ( r = 0.998) over a range of 1 to 10 000 TCID50/ml. The assay was specific to RV and its efficiency was confirmed by the detection of small amounts of viral RNA in clinical samples. Of the total 113 specimens, 80 (70%) and 76 (67%) were positive by real time RT-PCR and ELISA kit, respectively. The overall detection rate of the assay was not statistically different from that of ELISA ( χ^2 = 0.41, P 〉 0.5). Conclusion: The developed real time RT-PCR assay of group A rotavirus described here allows a large number of samples to be screened rapidly, sensitively and specifically, and it will potentially be a suitable tool for the detection of RV in field, samples including clinical specimen, polluted water, shellfish and so on.
出处 《山东大学学报(医学版)》 CAS 北大核心 2006年第3期217-221,共5页 Journal of Shandong University:Health Sciences
基金 国家"十五""食物安全关键技术"重大专项课题基金资助(2001BA804A22) 国家"十五"科技攻关计划基金资助课题(2003BA712A03-04)
关键词 实时逆转录聚合酶链反应 轮状病毒属 聚合酶链反应 Real time reverse transcriptase polymerase chain reaction Rotavirus Polymerase chain reaction
  • 引文网络
  • 相关文献

参考文献13

  • 1Mackay IM,Arden KE,Nitsche A.Real-time PCR in virology[J].Nucleic Acids Res,2002,30(6):1292-1305.
  • 2Castilho JG,Botelho MV,Lauretti F,et al.The in vitro cytopathology of a porcine and the simian (SA-11) strains of rotavirus[J].Mem Inst Oswaldo Cruz,2004,99(3):313-317.
  • 3Nonnenmacher C,Dalpke A,Mutters R,et al.Quantitative detection of periodontopathogens by real-time PCR[J].J Microbiol Methods,2004,59(1):117-125.
  • 4Exner MM,Lewinski MA.Sensitivity of multiplex real-time PCR reactions,using the LightCycler and the ABI PRISM 7700 Sequence Detection System,is dependent on the concentration of the DNA polymerase[J].Mol Cell Probes,2002,16(5):351-357.
  • 5Proudnikov D,Yuferov V,Zhou Y,et al.Optimizing primer-probe design for fluorescent PCR[J].J Neurosci Methods,2003,123 (1):31-45.
  • 6Schwarz BA,Bange R,Vahlenkamp TW,et al.Detection and quantitation of group A rotaviruses by competitive and real-time reverse-transcription polymerase chain reaction[J].J Virol Methods,2002,105(2):277-285.
  • 7Pang XL,Lee B,Boroumand N,et al.Increased detection of rotavirus using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay in stool specimens from children with diarrhea[J].J Med Virol,2004,72(3):496-501.
  • 8Poon LL,Chan KH,Wong OK,et al.Early diagnosis of SARS Coronavirus infection by real time RT-PCR[J].J Clin Virol,2003,28(3):233-238.
  • 9Ward CL,Dempsey MH,Ring CJA,et al.Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement[J].J Clin Virol,2004,29(3):179-188.
  • 10Mohamed N,Elfaitouri A,Fohlman J,et al.A sensitive and quantitative single-tube real-time reverse transcriptasePCR for detection of enteroviral RNA[J].J Clin Virol,2004,30(2):150-156.

同被引文献19

  • 1陈瑶,姚英民.轮状病毒肠道外感染的研究进展[J].医学综述,2006,12(3):145-147. 被引量:19
  • 2孙红,王斌,孙敏,刘瑜,朱军生,杨继红.免疫胶体金法与聚丙烯酰胺凝胶电泳检测儿童腹泻A组轮状病毒[J].检验医学,2006,21(6):679-681. 被引量:5
  • 3Pang XL, Lee B, Boroumand N, et al. Increased detection of rotavims using a real time reverse transcription - polymerase chain reaction ( RT -PCR) assay in stool specimens from children with diarrhea [ J]. J Med Virol, 2004,72 (3) :496 - 501.
  • 4Ferreira RG, Borges AM, Fiaccadori FS, et al.Gastroenteric virus detection in fecal samples from women in Goiania,State of Goias, Brazil[ J ].Rev Soc Bras Med Trop, 2010,43 (3) : 240-243.
  • 5Black RE,Cousens S,Johnson HL,et al.Global,regional,and national causes of child mortality in 2008:a systematic analysis[J].Lancet, 2010,375(9730) : 1969-1987.
  • 6Centers for Disease Control and Prevention (CDC).Rotavirus surveillance worldwide,2001-2008[J].MMWR Morb Mortal Wkly Rep, 2008,57 (46) : 1255-1262.
  • 7Ginzinger DG.Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream [J].Exp Hematol, 2002,30 ( 6 ) : 503- 512.
  • 8Kapikian AZ,Hoshino Y,Chanock RM.Rotaviruses [M].Knipe DM, HoMey PM.Fields Virology.4th ed.Philadelphia: Lippincott Willioms & Wilkins,2001.
  • 9尹兴晓,文喻玲,赵庆欢,于洋,陈元鼎.轮状病毒Vp6蛋白的免疫荧光检测[J].中国生物制品学杂志,2008,21(5):434-437. 被引量:7
  • 10陈华.婴幼儿轮状病毒腹泻的诊断与分析[J].中国妇幼保健,2009,24(14):1935-1936. 被引量:6

引证文献3

二级引证文献5

;
使用帮助 返回顶部