3rd International Workshop on HIV Drug Resistance 2-5 August 1994, Kauai, Hawaii, USA

A QUANTITATIVE POINT MUTATION ASSAY: PERFORMANCE PARAMETERS AND APPLICATION TO THE ASSAY OF GENOTYPIC DRUG RESISTANCE

Int Wkshop HIV Drug Res 1994 Aug 2-5;3:27 (abstract no. 26)

S. Kaye, C. Loveday, E. Comber and R.S. Tedder
Division of Virology, University College London Hospitals, UK

The association of point mutations of the HIV-1 genome with in vitro drug resistance is well established for many antiretroviral compounds. To examine the clinical significance of these mutations we have developed a PCR based quantitative point mutation assay (PMA). The assay is performed in a microtitre plate, allowing the analysis of large sample numbers and is able to determine the proportions of wild-type and mutant sequence at a given point. The assay has been in use in our laboratory for three years and has been used to detect and quantify 11 separate resistance associated point mutations of the RT gene in both cell-free virus RNA and proviral DNA extracts from clinical material. Point mutations are assayed in PCR product generated by nested PCR from proviral DNA or from cDNA reverse transcribed from RNA extracted from virus particles purified from clinical samples by immunocapture with anti-gpl20 coated latex. Each amplified sample is assayed in four streptavidin-coated microtitre wells by the addition of an oligonucletodie probe, Klenow polymerase and a single labelled dNTP. Relative proportions of the four bases are determined in a microtitre scintillation counter.

The assay has been adapted for the quantification of mutations associated with resistance to AZT (codons 41, 67, 70, 215, and 219), ddI (codon 74), 3TC (codon 184), ddC (codon 65) and non-nucleoside analogues (codon 181). Standard curves have been produced from prepared mixtures of wild-type and mutant plasmids or wild-type and mutant virus strains. The assay can consistently detect as little as 2% mutant sequence in a wild-type background. Reproducibility between duplicates was high (r=0.951, n=76) with highest variability being seen, as expected, where input copy number was low. The assay had been applied to the assay of mutations in large numbers of clinical samples in parallel with the assay of plasma viraemia by quantitative RT PCR. The samples assayed have been taken from subjects on open therapy and participants in controlled trials of both monotherapy and combination therapy. The assay fulfils the criteria needed for the monitoring of genotypic drug resistance in clinical material, is formatted to conveniently handle large numbers of samples and is easily adapted to the assay of many mutations.

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1994-08-02
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