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J Virol Methods. 2008 Nov;153(2):156-62. doi: 10.1016/j.jviromet.2008.07.020. Epub 2008 Sep 11.

Analysis of HCV resistance mutations during combination therapy with protease inhibitor boceprevir and PEG-IFN alpha-2b using TaqMan mismatch amplification mutation assay.

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  • 1Virology, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA. stephanie.curry@spcorp.com

Abstract

TaqMan Mismatch Amplification Mutation Assay (TaqMAMA) is a highly sensitive allelic discrimination method. The mismatch amplification mutation assay (MAMA) is based on preferential amplification of mutant allele by the 'MAMA' primer, which is designed to have two mismatches with the wild-type allele and only one mismatch with the mutant allele. In this report, the TaqMAMA method was adapted for the detection and quantitation of minor HCV variants resistant to the protease inhibitor boceprevir (SCH 503034) from clinical samples. A good correlation of mutant frequency was observed between TaqMAMA and the results of clonal sequencing. TaqMAMA detected consistently minor variants at a level as low as 0.1%. Using TaqMAMA, it was demonstrated that resistant variants existed in the viral population before boceprevir treatment. The frequency of two resistant mutants (T54A and V170A) increased significantly during treatment with boceprevir, but was suppressed by combination treatment of PEG-IFN alpha-2b and boceprevir. The prevalence of both mutants decreased at the end of the two-week follow-up period. These results show that TaqMAMA can be used to detect minor resistant variants in pretreatment samples and to study in detail the evolution of mutant viruses during targeted antiviral therapy.

PMID:
18755220
[PubMed - indexed for MEDLINE]
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