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Quantification of HIV-1 using multiple quantitative polymerase chain reaction standards and bioluminometric detection
KTH, Superseded Departments, Biochemistry and Biotechnology.
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2001 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 288, no 1, 28-38 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2001. Vol. 288, no 1, 28-38 p.
Keyword [en]
competitive, polymerase chain reaction, quantification, multiple quantitative standards, bioluminescence, hepatitis-b virus, competitive pcr, viral load, colorimetric detection, type-1 rna, dna, infection, plasma, assay, amplification
URN: urn:nbn:se:kth:diva-20279ISI: 000166328800004OAI: diva2:338973
QC 20100525Available from: 2010-08-10 Created: 2010-08-10Bibliographically approved

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