期刊文献+

非洲马瘟病毒实时荧光定量RT-PCR检测方法的建立 被引量:4

Development of Real-time Fluorescent Quantitative RT-PCR for Detection of AHSV Based on SYBR GreenⅠ
下载PDF
导出
摘要 非洲马瘟病毒(AHSV)为双股RNA病毒,能感染所有马科动物。为建立一种AHSV快速检测方法,根据GenBank及作者所测AHSV 9个血清型VP7-ORF全长核苷酸序列,设计一对位于AHSV基因组S7片段的特异引物,然后进行荧光定量检测。经试验,证实该对引物对9种血清型的AHSV RNA均有特异性扩增,能检出特异荧光信号,而对与AHSV同属的蓝舌病病毒(BTV)、鹿流行性出血症病毒(EHDV)及正常马淋巴组织和阴性对照均无扩增,无有效荧光信号可检出。以国家外来动物疫病诊断实验室已构建好的AHSV 9型pMD18-T-VP7质粒为标准品可建立良好的标准曲线,实现对检测样本的实时定量。本研究建立了模板预变性结合"三步"法敏感特异的AHSV实时荧光定量RT-PCR快速检测方法,检测灵敏度可达102拷贝/μL。 African horse sickness virus(AHSV)is a double-stranded ribonucleic acid(dsRNA)virus which can infect all the equid animals.Based on the VP7-ORF sequences from GenBank and results of sequencing for nine serotypes of AHSV previously completed by our own team,apair of specific primers located on the segment S7 were designed for SYBR GreenⅠquantitative detection,in order to develop one kind of quick method for detecting the AHSV.Results showed that the primer pair was identified to be specific for each of the 9 serotypes of AHSV only and specific fluorescent signal could be detected,but not for the related viruses including BTV,EHDV and normal lymphoid tissue of horse without specific fluorescent signal.The built positive recombinant plasmid(AHSV9pMD-18T-VP7)in our lab was used for a positive quantitative template to establish a standard curve.Finally,the method of pre-denaturalized template based on"three-step"reaction was established in the study.It was demonstrated that the SYBR GreenⅠfluorescent quantitative RT-PCR for detection of AHSV could be used for the rapid diagnosis and quantitative detection with detection limit of 102 copies/μL cDNA.
出处 《动物医学进展》 CSCD 北大核心 2013年第12期11-16,共6页 Progress In Veterinary Medicine
基金 公益性行业(农业)科研专项(200903037)
关键词 非洲马瘟病毒 血清型 SYBR Green 实时荧光定量RT-PCR AHSV serotype SYBR GreenⅠ real-time fluorescent quantitative RT-PCR
  • 相关文献

参考文献9

  • 1OIE. Manual o{ diagnostic tests and vaccines for terrestrial an- imals (mammals, birds and bees) [Z]. 2008 : 823-838.
  • 2Alexander K A, Kat P W, House J, et al. African horse sick- ness and African carnivores[J]. Vet Mierobiol, 1995, 47:133-140.
  • 3Sonja M, Janusz T. Preparation of recombinant African horse sickness virus VP7 antigen via a simple method and validation of a VP7-based indirect ELISA for the detection of group-spe- cific IgG antibodies in horse sera [J]. J Virol Meth, 2005, 125, 55-65.
  • 4赵文华,杨仕标,王金萍,李富祥.非洲马瘟病毒群特异性RT-PCR检测方法的研究[J].生物技术通报,2011,27(4):204-207. 被引量:14
  • 5赵文华,杨仕标,王金萍,李富祥.非洲马瘟病毒分型RT-PCR检测方法的研究[J].中国农学通报,2012,28(2):43-47. 被引量:7
  • 6Rodriguez M, Hooghuis H, Castano M. Current status of the diagnosis and control of African horse sickness[J]. Vet Res, 1993,24:189-197.
  • 7Imadeldin E A. PCR detection of African horse sickness virus serogroup based on genome segment three sequence analysis [J]. J Virol Meth,2009,159:1-5.
  • 8Bustin S A. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays[J]. J Mol Endocrinol, 2000,25 (2) : 169-193.
  • 9Vandesompele J, De P A , Speleman F. Elimination of primer dimer artifacts and genomic coamplification using a two-step SYBR green I real-time RT-PCR[J]. Anal Biochem,2002, 303 (1) : 95-98.

二级参考文献23

  • 1曾昭文,花群义,段纲,项勋,常华.非洲马瘟研究进展[J].中国农学通报,2006,22(9):17-22. 被引量:5
  • 2Oellerman R A, Els H J, Erasmus B J. Characterisation of African horse sickness virus[J]. Arch Gesamte Virus for sch,1970,29: 163-174.
  • 3OIE. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals(Mammals,Birds and Bees).2008:823-838.
  • 4Alexander K A, Kat P W, House J, et al. African horse sickness and African carnivores[J]. Veterinary Microbiology, 1995, 47: 133-140.
  • 5Fernandez P J, Ferrandez P P, Rodriguez B, et al. Rapid and sensitive detection of Afrlean horse sickness virus by real-time PCR [J]. Research in Veterinary Science,2009,86:353-358.
  • 6Rodriguez S B, Fernandez P J, Sailleau C, et al. Novel gel-based and real-time PCR assays for the improved detection of African horse sickness virus[J]. Journal of Virological Methods,2008,151: 87-94.
  • 7Imadeldin E A. PCR detection of African horse sickness virus serogroup based on genome segment three sequence analysis[J]. Journal of Virological Methods,2009,159:1-5.
  • 8Sailleau C, Moulay S, Cruciere C, et al. Detection of African horsesickness virus in the blood of experimentally infected horses: comparison of virus isolation and a PCR assay[J]. Research in Veterinary Science, 1997,62:229-232.
  • 9Zientara S, SaiUeau C, MouLay S, et al. Application of the polymerase chain reaction to the detection of African horse sickness viruses[J]. Journal of Virological Methods,1995,53:47-54.
  • 10Sailleau C, Hamblin J, Paweska T, et al. Identification and differentiation of the nine African horse sickness virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2 [J]. Journal of General Virology,2000,81:831-837.

共引文献13

同被引文献43

引证文献4

二级引证文献5

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部