New estimation method for highly sensitive quantitation of human immunodeficiency virus type 1 DNA and its application.

Abstract A new estimation method for quantitation of HIV-1 DNA was established by introducing a pre-quantitation polymerase chain reaction (PCR) before conventional real-time PCR. Two alternative methods for estimating the copy number can be used: the first method utilizes the rate of β2-microglobulin (β2M) gene amplification during the pre-quantitation PCR, and the second utilizes a calibration curve of the crossing point of real-time PCR versus the standard HIV-1-plasmid concentration. These methods could be used to reproducibly and accurately detect a provirus density down to five copies/10 6 cells (for methods 1 and 2, inter-assay CV = 17 and 16% and accuracy = 81 and 92%, respectively). The levels of HIV-1 DNA could be measurable using as little as 100 μl of whole blood or buffy coat cells. Using a combination of a conventional and highly sensitive methods, we found that the amount of HIV-1 DNA ranged from 2 to 5960 copies/10 6 cells (median of 830 copies/10 6 cells) in CD4-positive T lymphocytes isolated from 30 patients responding well to highly active antiretroviral therapy (HAART). Thus, the highly sensitive method developed in this study allows estimation of the HIV-1 reservoirs in peripheral CD4-positive T lymphocytes of patients responding well to HAART.