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应用Taqman MGB定量PCR技术建立HTLV-I前病毒的检测体系

Quantitation of HTLV-I Proviral Load Using Real-time Quantitative PCR with Taqman MGB Probe

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【作者】 谢金镇陈长荣张军倪宏英葛胜祥周娟娟欧山海郑秀娟冉鹏裴斌

【Author】 XIE Jin-zhen1,CHEN Chang-rong1,ZHANG Jun2,NI Hong-ying1,GE Sheng-xiang2,ZHOU Juan-juan1,OU Shan-hai1,ZHENG Xiu-juan1,RAN Peng1,PEI Bin1(1.Xiamen Blood Services,Xiamen 361004,China;2.National Institute of Diagnostics and Vaccine Development in Infectious Diseases,Xiamen University,Xiamen 361005,China)

【机构】 厦门市中心血站厦门大学国家传染病诊断试剂与疫苗工程技术研究中心

【摘要】 建立了一种快速检测HTLV-I前病毒的荧光定量方法。利用HTLV前病毒DNA序列设计特异性TaqmanMGB探针及引物对目标DNA片段进行实时检测,用含目标片段质粒构建标准曲线。该定量检测方法在103~107copy/μL范围内与Ct值呈良好线性关系(R2可达0.999),最低检测浓度为5copy/μL。利用该系统对献血者血液标本进行HTLV前病毒初筛确认及定量检测,结果表明该方法重复性良好,特异性高,适用于献血者和临床标本的检测和确认,亦可用于对HTLV病毒载量与成人T细胞白血病等疾病的关联性进行研究。

【Abstract】 A quantitative real-time PCR assay was developed to measure the proviral load of human T-lymphotropic virus type I(HTLV-I) in peripheral blood.The technology utilizes special primers and Taqman MGB fluorescence probe to measure amplification products from the gag-pro-pol polyprotein gene of HTLV-I.HTLV-I copy number was normalized to the amount of cellular DNA by quantitation of the β-actin gene.The amplification system was sensitive to detect 5copy/μL.The standard curve had a good linearity when the quantity for the gene was between 103 and 107 copy/μL(R2=0.999).Good reproducibility was observed in each intra-and inter-assay.We also measured proviral load in peripheral blood in 12 HTLV-I seropositive former blood donors.Proviral load for HTLV-I infected donors ranged from 0.015 to 12.819 copy/cell in WBC with the mean of 3.116 copy/cell.

  • 【文献出处】 病毒学报 ,Chinese Journal of Virology , 编辑部邮箱 ,2009年05期
  • 【分类号】R450
  • 【被引频次】4
  • 【下载频次】214
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