Infection By Hiv-1 Pcr And Quantitative Methods Of The Structure Of The Hiv Core Protein Gene Analysis And Cloning

The pathogenesis of AIDS is still unclear up to now. The high degree of genetic variations made it very difficult to cure the serious disease. All the therapeutic strategies were unsatisfactory until now. In order to evaluate the efficacy of various treatment measures objectively and to explore the mechanism of AIDS development we applied molecular cloning, PCR and other modern molecular biological techniques etc in our studies to detect the pathogenic agent-HIV-1. We developed a very useful method to quantify the virus load in peripheral blood mononuclear cells. Also we performed molecular epidemiological analysis in this study.First we used recombinant PCR to generate an internal standard as competitive template. A region in the HIV-1 gag gene, highly conserved among divergent viral isolates, was selected as our target sequence. A plasmid containing this sequence (PCR II gag) and a plasmid identical to PCR II gag (PCR II Δ gag), except for an internal 50 bp deletion, were generated as a positive control target sequence and competitive template. Gradient dilutions of competitive template were added to and coamplified with the test sample template in different reaction cocktail. By this way we developed a competitive PCR (QC- PCR) method for quantitation of HIV-1 provirus DNA. 8 samples of peripheral blood mononuclear cells (PBMC) from AIDS patients were prepared by urea-SDS lysis buffer, phenol/chloroform and ethanol and performed by QC-PCR reaction. In addition we coamplified the single copy sequence gene, β -Globin gene,with HIV-1 gag gene and developed differential PCR to detect the HIV-1 nucleic acids in these PBMC samples. The results of these two method were quite similar. They were very suitable for analysis of clinical specimens and useful in many other fields.In order to find the individual with HIV infection quickly and easily we detected the HIV-1 DNA in dried blood spot samples.Two steps of treatment for this kind of samples and PCR amplification were used and we got a very strong PCR signal. It suggests that the DBS is useful to detect the virus DNA. Also 26 serum samples were obtained mainly from Yunnan province, the southwest part of China. By PCR-SSCP and HA analysis we determined the major type of SSCP of HIV-1 in this area. The specific PCR product was cloned into pUCl9 vector digested by Sma I. Several kinds of method to identify the cloned gag gene were carried out and the result showed which is correct. The successful cloning of HIV-1 gag gene seems promising for sequencing in the future.