Development and assessment of a multiplex real-time PCR assay for quantification of human immunodeficiency virus type 1 DNA
| Autor: | Apostolos Beloukas, Angelos Hatzakis, Vana Sypsa, C. Haida, Dimitrios Paraskevis |
|---|---|
| Rok vydání: | 2009 |
| Předmět: |
Microbiology (medical)
virus diseases HIV Infections Biology Viral Load Virology Molecular biology Peripheral blood mononuclear cell Polymerase Chain Reaction Sensitivity and Specificity Virus law.invention chemistry.chemical_compound Real-time polymerase chain reaction chemistry law Molecular beacon Multiplex polymerase chain reaction DNA Viral HIV-1 Humans Multiplex Polymerase chain reaction DNA |
| Zdroj: | Journal of clinical microbiology. 47(7) |
| ISSN: | 1098-660X |
| Popis: | Previous studies showed that high levels of human immunodeficiency virus type 1 (HIV-1) DNA are associated with a faster progression to AIDS, an increased risk of death, and a higher risk of HIV RNA rebound in patients on highly active antiretroviral therapy. Our objective was to develop and assess a highly sensitive real-time multiplex PCR assay for the quantification of HIV-1 DNA (RTMP-HIV) based on molecular beacons. HIV-1 DNA quantification was carried out by RTMP in a LightCycler 2.0 apparatus. HIV-1 DNA was quantified in parallel with CCR5 as a reference gene, and reported values are numbers of HIV-1 DNA copies/10 6 peripheral blood mononuclear cells (PBMCs). The clinical sensitivity of the assay was assessed for 115 newly diagnosed HIV-1-infected individuals. The analytical sensitivity was estimated to be 12.5 copies of HIV-1 DNA per 10 6 PBMCs, while the clinical sensitivity was 100%, with levels ranging from 1.23 to 4.25 log 10 HIV-1 DNA copies/10 6 PBMCs. In conclusion, we developed and assessed a new RTMP-HIV assay based on molecular beacons, using a LightCycler 2.0 instrument. This multiplex assay has comparable sensitivity, reproducibility, and accuracy to single real-time PCR assays. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|