New estimation method for highly sensitive quantitation of human immunodeficiency virus type 1 DNA and its application.
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 beta2-microglobulin (beta2M) 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 microl 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.
Nagai
Hiromi
H
Clinical Research Center, National Hospital Organization Nagoya Medical Center, 4-1-1 Sannomaru, Naka-ku, Nagoya, Aichi 460-0001, Japan.
Wada
Kaoru
K
Morishita
Takayuki
T
Utsumi
Makoto
M
Nishiyama
Yukihiro
Y
Kaneda
Tsuguhiro
T
D000163:Acquired Immunodeficiency Syndrome / Q000188:drug therapy / Q000821:virology; D023241:Antiretroviral Therapy, Highly Active; D001483:Base Sequence; D015496:CD4-Positive T-Lymphocytes / Q000821:virology; D004279:DNA, Viral / Q000032:analysis* / Q000097:blood; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D012189:Retrospective Studies; D012680:Sensitivity and Specificity
D004279:DNA, Viral