Quantification of HIV-1 using multiple quantitative polymerase chain reaction standards and bioluminometric detection.
A non-gel-based quantification assay based on competitive PCR and bioluminometric detection has been developed. Samples containing human immunodeficiency virus type 1 (HIV-1) DNA and three quantitative standards at discrete concentrations were coamplified by PCR with primers annealing in the polymerase gene region. The quantitative standards contained the same primer binding sequences and had the same amplicon length as the wild-type DNA, but differed in an internal homopolymeric stretch (A, C, or T) over three base pairs. The PCR products were captured onto a solid support and treated with NaOH to separate the strands. Discrimination between the wild-type DNA and the three quantitative standard amplicons was achieved on the solid support by four parallel extension reactions with 3'-end specific primers. Inorganic pyrophosphate (PPi) released as a result of successful extension was converted to ATP by ATP sulfurylase and the level of ATP was sensed by firefly luciferase, generating a proportional amount of visible light which was detected by a luminometer. Here, we show that the obtained calibration curves, using the signal intensities of the three quantitative standards, enabled determination of the amount of target HIV-1 DNA.
Nygren
M
M
Department of Biotechnology, Royal Institute of Technology (KTH), Stockholm, SE-100 44, Sweden.
Ronaghi
M
M
Nyrén
P
P
Albert
J
J
Lundeberg
J
J
D001483:Base Sequence; D001667:Binding, Competitive; D002138:Calibration; D017931:DNA Primers; D018628:Gene Dosage; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D008163:Luminescent Measurements; D009838:Oligodeoxyribonucleotides; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012367:RNA, Viral / Q000032:analysis*; D012015:Reference Standards
D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D012367:RNA, Viral