Alu-LTR PCR检测整合型HIV-1实验方法的优化

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。

Optimization of Alu-long Terminal Repeat Polymerase Chain Reaction Approach to Detect Integrated HIV-1

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.