| 扩增潜伏HIV-1 DNA序列实验方法的优化 |
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| 引用本文： | 王鹏飞,焦艳梅,朱焕章,吴昊.扩增潜伏HIV-1 DNA序列实验方法的优化[J].北京医学,2010,32(12):972-974. |
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| 作者姓名： | 王鹏飞 焦艳梅 朱焕章 吴昊 |
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| 作者单位： | 上海,复旦大学生命科学学院遗传学研究所,遗传工程国家重点实验室200433;首都医科大学附属北京佑安医院感染科 |
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| 基金项目： | 国家"十一五"传染病重大专项 |
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| 摘 要： | 目的对PCR扩增潜伏HIV-1DNA序列的实验方法进行优化。方法收集30例经高效抗逆转录病毒治疗（HAART）6~12个月的HIV-1感染者抗凝血标本,分别从外周血单个核细胞（PBMC）和高度纯化的CD4＋T细胞提取基因组DNA,巢式PCR扩增HIV-1env的C2-V5区,电泳检测扩增产物并进行DNA序列测定。结果同一患者PBMC纯化CD4＋T细胞标本扩增HIV-1env基因C2-V5区的效率（93.3%）明显高于直接从PBMC标本扩增该目的片段的效率（10.0%）。结论 PBMC纯化CD4＋T细胞后,再提取基因组DNA进行巢式PCR扩增,可提高扩增潜伏HIV-1DNA序列的效率。
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| 关 键 词： | 潜伏HIV-1 DNA 巢式PCR CD4+T细胞 外周血单个核细胞 |
Optimization of approach to amplify latent HIV-1 DNA sequence |
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| Institution: | WANG Peng-fei,JIAO Yan-mei,ZHU Huan-zhang,et al(State Key Laboratory of Genetic Engineering,Institute of Genetics,School of Life Sciences,Fudan University,Shanghai 200433) |
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| Abstract: | Objective To optimize polymerase chain reaction（PCR） approach to amplify latent HIV-1 DNA sequence.Methods Thirty anticoagulation blood samples from HIV-1 infection,with HAART treatment for 6-12 months were obtained.The C2-V5 region of HIV-1 env gene was amplified by nested PCR from DNA extraction of PBMC or highly purified CD4＋T lymphocytes respectively.Then the amplified products were tested by agarose gel electrophoresis and DNA sequencing.Results The efficiency of amplifying the C2-V5 region of HIV-1 env gene from purified CD4＋T lymphocytes was significantly higher than that from PBMC.Conclusion Optimized scheme of amplifying latent HIV-1 DNA sequence should be purify CD4＋T lymphocytes from PBMC then extract genomic DNA and amplify by nested PCR. |
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| Keywords: | Latent HIV-1 DNA Nested PCR CD4＋T lymphocytes PBMC |
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