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目的建立Alu-反向PCR检测整合型HIV-1DNA的实验方法。方法收集18例HIV-1感染者及12例健康者的抗凝血标本,根据整合型HIV-1DNA的特点设计引物,第一轮PCR5’端引物来自于人类保守的Alu序列,3’端引物来自于HIV-1gag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-1DNA序列。经PstⅠ酶切后进行自身片段环化,以此为模板设计针对HIV-1LTR和gag保守区域引物,进行巢式PCR。结果 16例HIV-1感染者成功扩增出目的片段,12例健康者均为阴性。结论本实验建立的检测整合型HIV-1的方法为进一步研究HIV病毒储藏库机制提供了参考。
Objective To establish an Alu-reverse PCR method for the detection of integrated HIV-1 DNA. Methods The anticoagulant samples of 18 HIV-1 infected individuals and 12 healthy controls were collected. Primers were designed according to the characteristics of the integrated HIV-1 DNA. The first round of PCR 5 ’primer was from the human conservative Alu sequence and the 3’ From the HIV-1 gag sequence, the amplified fragment contains the human genomic DNA sequence upstream of the integration site and the integrated HIV-1 DNA sequence. After digestion with PstI, the fragment was self-circularized and used as a template to design primers for HIV-1 LTR and gag conserved regions for nested PCR. Results 16 HIV-1 infected patients successfully amplified the target fragment, 12 healthy people were negative. Conclusion The method of detecting integrated HIV-1 established in this experiment provides a reference for further research on the mechanism of HIV virus storage.