Discussion The PCR Detection Methods Of HIV-1 Reservoir And Clinical Significance In Peripheral Blood PBMC

Objective:To evaluate the reliability and specificity of the two methods detecting the size of HIV-1 reservoir,and to select which is more suitable for clinical application.And verify the reliability and practicability of the selected method with a cross-sectional sample.Methods:(1)Total DNA was extracted from peripheral blood mononuclear cells of HIV-1 infected patients using QIAamp DNA Mini kit;(2)Alu-PCR was used to verify the specificity of HIV-1 integrated DNA using two sets of primer regimens(3)To verify the sensitivity and specificity of HIV-1 total DNA by fluorescence probe PCR,And further assess the reliability and practicability of the PCR assay for quantification of total HIV-1 DNA by using a sample of HIV-1 infected individuals with cross-sectional HIV-1 RNA negative for 1 to 6years.Results:(1)Alu-PCR was used to detect integrated HIV-1 DNA:The DNA samples from PBMCs of 20 HIV-1 infected patients were free of bands in the electrophoresis patterns of the first round of PCR products in Scheme 1 and Scheme 2,and the amplification products were low and the gene sequencing could not be completed.In the second round of PCR amplification products,there were 9 cases(n=20)in the program 1 with bright bands at 240bp,and the amplified products were sequenced and sequenced.Four of them were consistent with the integrated HIV-1 DNA fragments,5 cases only human genome fragments,The positive rate of HIV-1 DNA fragment integration in the amplified product was 20%(4/20).;12 cases(n=20)in program 2 hadbright band at 350bp,the amplified products were sequenced after sequence alignment,there are HIV-1 DNA fragments without Human genome fragments,The positive rate of HIV-1 DNA fragment in the amplified product was 60%(12/20)?(2)In our study,the range of total HIV-l DNA quantitatiion were50-10~4??/10~6cells;The positive rate of HIV-1 total DNA was 100%in 20cases of HIV-1 infected PBMC DNA digestion kit,The amplified products were sequenced by gene sequencing,and the similarity with HIV-1 DNA was more than 90%.The positive rate of HIV-1 total DNA in 8 healthy controls was 0,The experimental specificity was 100%.47 cases of cross-sectional clinical samples were detected by this method,The mean total HIV-1 DNA of untreated HIV-1 infected patients was(3.24±0.58)log10 copies/10~6 PBMC,while those patiens were(2.93±0.69)?(2.84±0.55)?(2.53±0.48)log10 copies/10~6 PBMC respectively after HIV-1 RNA is below the detection limit of 1-2 years?3-4years?5-6years.The overall trend is that the longer the HIV-1 RNA turn native,the lower the HIV-1 total DNA is.Conclusion:(1)the specificity of alu-PCR detecting HIV-1 integrated DNA should be further verified.(2)Fluorescence probe PCR detection of total HIV DNA method,high detection rate,high sensitivity,feasibility and practicality,is expected to become the serum markers for consumption of HIV-1 reservoir.