Open AccessDevelopment and Assessment of a Multiplex Real-Time PCR Assay for Quantification of Human Immunodeficiency Virus Type 1 DNA
Author(s) -
Apostolos Beloukas,
Dimitrios Paraskevis,
C. Haida,
Vana Sypsa,
Angelos Hatzakis
Publication year - 2009
Publication title -
journal of clinical microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.349
H-Index - 255
eISSN - 1070-633X
pISSN - 0095-1137
DOI - 10.1128/jcm.01264-08
Subject(s) - multiplex , peripheral blood mononuclear cell , molecular beacon , real time polymerase chain reaction , virology , biology , polymerase chain reaction , lentivirus , dna , viral load , multiplex polymerase chain reaction , virus , microbiology and biotechnology , immunology , viral disease , gene , in vitro , bioinformatics , genetics , oligonucleotide
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.