【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
【机 构】
：
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,首都医科大学附属北京佑安医院感染科,
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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。
Objective To optimize the experimental method for PCR amplification of latent HIV-1 DNA sequence. Methods A total of 30 anticoagulant samples of HIV-1 infected patients were collected from HAART patients for 6 ~ 12 months. Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs) and highly purified CD4 + T cells DNA was amplified by nested PCR from C2-V5 region of HIV-1 env. The amplification product was detected by electrophoresis and the DNA sequence was determined. Results The efficiency (93.3%) of amplification of HIV-1 env gene C2-V5 region from PBMC-purified CD4 + T cells in the same patients was significantly higher than that of the PBMC samples (10.0%). Conclusion PBMC purified CD4 + T cells, and then extracted genomic DNA for nested PCR amplification can increase the efficiency of amplification of latent HIV-1 DNA sequence.
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