Quantification of HIV-1 using multiple competitors in a single-tube assay.
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.
Vener
T
T
KTH, Royal Institute of Technology, Swedish Institute for Infectious Disease Control, Stockholm, Sweden.
Axelsson
M
M
Albert
J
J
Uhlén
M
M
Lundeberg
J
J
D001483:Base Sequence; D001667:Binding, Competitive; D002138:Calibration; D017931:DNA Primers; D015658:HIV Infections / Q000097:blood / Q000821:virology; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D014766:Viremia / Q000821:virology
D017931:DNA Primers