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Alu-LTR PCR检测整合型HIV-1实验方法的优化 被引量：2

Optimization of Alu-long Terminal Repeat Polymerase Chain Reaction Approach to Detect Integrated HIV-1
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摘要 目的对Alu-LTRPCR检测整合型HIV-1的实验方法进行优化。方法收集20例HIV-1感染患者及10例健康对照者的抗凝血标本,纯化CD4+T细胞并提取DNA,根据整合型HIV-1DNA的特点设计引物,首轮PCR5′端引物来自于人类保守的Alu序列,3′端引物来自于HIV-1LTRU5区序列,扩增片段包含了整合位点上游的人类基因组DNA序列和整合的HIV-1LTR的序列。第二轮引物针对HIV-1LTRU3区的序列。在首轮PCR基础上,PCR产物稀释后进行第二轮PCR。结果经过优化的Alu-LTRPCR,20例HIV病人全部检测到整合的HIV-1。结论经优化后,Alu-LTRPCR检测整合型HIV-1的实验方法的灵敏度明显提高。 Objective To optimize Alu-long terminal repeat （LTR） polymerase chain reaction （PCR） approach to detect integrated HIV-1. Methods The genomic DNA from purified CD4^＋ T cells of 20 HIV-1 patients and 10 healthy control subjects was obtained. A pair of primers were designed according to the characteristics of human genome and HIV-1 genome. The first PCR was carried out by using the nested 5＇ primers from conserved sequences of human Alu and 3＇ primer from conserved HIV-1 long terminal repeat （LTR） sequences. The product of the first PCR was representative of both cellular DNA upstream of the integration site and integrated HIV-1 LTR. An aliquot of the diluted first PCR product was further subjected to the second round of PCR by using nested HIV-1 LTR- specific primers and then detect the DNA sequence from the product of Alu-LTR PCR. Results After optimized Alu-LTR PCR, integrated HIV-1 was detected in each of the 20 cases of HIV/AIDS. Conclusion After optimization, the sensitivity of Alu-LTR PCR for detection of integrated HIV-1 was markedly improved.
作者 李娟 焦艳梅 画伟 张彤 吴昊 高艳青
机构地区 首都医科大学附属北京佑安医院性病艾滋病诊疗中心
出处 《首都医科大学学报》 CAS 北大核心 2009年第5期631-634,共4页 Journal of Capital Medical University
基金 国家"十一五"传染病重大专项(2008ZX10001-006) 国家自然科学基金(30872226)资助项目~~
关键词 Alu-PCR 整合型HIV-1 CD4+T细胞 Alu-polymerase chain reaction integrate human immunodeficiency virus-1 CD4 ^＋ T cell
分类号 R512.91 [医药卫生—内科学]
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参考文献12

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共引文献4

同被引文献19

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首都医科大学学报
2009年 第5期

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