Open AccessQuantification of HIV-1 Using Multiple Competitors in a Single-Tube Assay
Author(s) -
Tanya Vener,
Maria Axelsson,
Jan Albert,
Mathias Uhlén,
Joakim Lundeberg
Publication year - 1996
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/96212st01
Subject(s) - primer (cosmetics) , dna , biology , microbiology and biotechnology , human immunodeficiency virus (hiv) , polymerase chain reaction , calibration curve , dna sequencer , dna sequencing , genetics , virology , chemistry , chromatography , gene , detection limit , organic chemistry
Methods for quantification of human immunodeficiency virus type 1 (HIV-1) based on competitive PCR and fragment analysis have been developed. Samples containing HIV-1 DNA and known amounts of three cloned competitors were co-amplified by PCR with semi-nested primers. The competitor DNAs contained the same long terminal repeat primer binding sequences as the wild-type DNA, but they are different in internal sequences and length. One of the inner primers was fluorescent-labeled to allow discrimination between the wild-type DNA and the three competitors by fragment analysis using a standard automated sequencer. A calibration curve using the peak area of the three competitors enabled accurate determination of target amount with minimal variations. The method presented here can be used for quantification of HIV-1 in clinical samples and will be useful for monitoring disease progression and treatment effects.