Development and Assessment of a Multiplex Real-Time PCR Assay for Quantification of Human Immunodeficiency Virus Type 1 DNA

• Published in: Journal of Clinical Microbiology Vol. 47; no. 7; pp. 2194 - 2199
• Main Authors: Beloukas, A, Paraskevis, D, Haida, C, Sypsa, V, Hatzakis, A
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.07.2009; American Society for Microbiology (ASM)
• Subjects: Biological and medical sciences; DNA, Viral - blood; DNA, Viral - genetics; Fundamental and applied biological sciences. Psychology; HIV Infections - virology; HIV-1 - genetics; HIV-1 - isolation & purification; Human immunodeficiency virus 1; Human viral diseases; Humans; Infectious diseases; Medical sciences; Microbiology; Miscellaneous; Polymerase Chain Reaction - methods; Sensitivity and Specificity; Viral diseases; Viral diseases of the lymphoid tissue and the blood. Aids; Viral Load - methods; Virology; Immunopathology; Microbiology; Multiplex polymerase chain reaction; HIV-1 virus; Retroviridae; AIDS; Real time; Immune deficiency; Lentivirus; Infection; Virus; Viral disease; Human immunodeficiency virus; Quantitative analysis
• ISSN: 0095-1137; 1098-660X
• DOI: 10.1128/JCM.01264-08

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/10⁶ 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 10⁶ PBMCs, while the clinical sensitivity was 100%, with levels ranging from 1.23 to 4.25 log₁₀ HIV-1 DNA copies/10⁶ 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.