Application of the fluorescence quantitative method to detect the integrated HIV DNA in the clinical study
10.3760/cma.j.issn.1003-9279.2017.06.019
- VernacularTitle: 整569合型HIV DNA荧光定量检测法的临床初探
- Author:
Min ZHANG
1
;
Qianying WANG
1
;
Xin LI
1
;
Yunwen HU
1
;
Bisheng SHI
2
Author Information
1. Department of Clinical Laboratory, Scientific Research Unit
2. Shanghai Public Health Clinical Center, Shanghai 201508, China
- Publication Type:Journal Article
- Keywords:
HIV;
Fluorescence quantitative PCR;
ACH2 cell;
CD3 gene
- From:
Chinese Journal of Experimental and Clinical Virology
2017;31(6):566-569
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish fluorescence quantitative polymerase chain reaction (PCR) to detect integrated HIV DNA in peripheral blood mononuclear cells.
Methods:A total of 30 HIV-seropositve individuals were enrolled in this study, including 10 subjects with a detection limit of 20 copies/ml of plasma, 10 patients with drug resistance and 10 patients with no history of antiretroviral therapy (ART). Cultivated ACH2 cells carried a single copy of the integrated HIV genome. We have built pMD19T-CD3 plasmid and calculated the copy number. We used oligonucleotides ULF1 specific for the long terminal repeats (LTR) regions and two oliligonucleotides specific for human Alu sequences to pre-amplified the integrated HIV DNA. Samples and serial dilutions of ACH2 cells were all pre-amplified, the products of which were used for the second round fluorescence amplifications. The Lambda T primers, UR2 primers and HIV Taqman probes were used for second round amplifications in integrated HIV DNA assay. The CD3IN5 primers, CD3IN3 primers and CD3 Taqman probes were used for CD3 quantification.
Results:Serial 5-fold dilutions of the plasmid were used as standards for CD3 gene quantifications. The equation of the linear regression was y=-2.731x+ 43.01(R2=0.953). The cellular input was quantified by number of human genome equivalents (CD3 gene, 2 copies/cell). The copies of ACH2 cells was 4.271×104, which was the cellular input of the ACH2 cells. Serial 5-fold dilutions of ACH2 cells were used to generate a standard curve for the integrated HIV DNA assays. The equation of the linear regression was y=-3.146x+ 39.11 (R2=0.968). The ratio between the number of copies of the integrated HIV DNA form and the number of cells (CD3 copies) was calculated to obtain the frequency of cells. Based on the test between different groups, each group had no difference (P> 0.05).
Conclusions:This method presented advantage in detection of the lower copies of virus. It hinted that the current antiretroviral therapy can not effectively attack against latent viral reservoirs. It also provided a basis for new therapeutic intervention.
