New estimation method for highly sensitive quantitation of human immunodeficiency virus type 1 DNA and its application
| Autor: | Tsuguhiro Kaneda, Takayuki Morishita, Kaoru Wada, Hiromi Nagai, Makoto Utsumi, Yukihiro Nishiyama |
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| Rok vydání: | 2005 |
| Předmět: |
CD4-Positive T-Lymphocytes
Molecular Sequence Data Buffy coat Biology Sensitivity and Specificity Virus law.invention chemistry.chemical_compound law Antiretroviral Therapy Highly Active Virology Gene duplication Humans Polymerase chain reaction Retrospective Studies Whole blood Acquired Immunodeficiency Syndrome Base Sequence Provirus Molecular biology Real-time polymerase chain reaction chemistry DNA Viral HIV-1 DNA |
| Zdroj: | Journal of Virological Methods. 124:157-165 |
| ISSN: | 0166-0934 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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