Quantification of HIV-1 using multiple competitors in a single-tube assay
Vener, T.; Axelsson, M.; Albert, J.; Uhlén, M.; Lundeberg, J.
Biotechniques 21(2): 248-52 253-5
1996
ISSN/ISBN: 0736-6205 PMID: 8862809 Document Number: 459456
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.