1. Detection and quantification of minority HIV isolates harbouring the D30N mutation by real-time PCR amplification.
- Author
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Detsika MG, Chandler B, Khoo SH, Winstanley C, Cane P, Back DJ, and Owen A
- Subjects
- HIV drug effects, Humans, Nelfinavir pharmacology, Sensitivity and Specificity, Amino Acid Substitution genetics, Drug Resistance, Viral genetics, HIV genetics, HIV Infections virology, Mutation, Polymerase Chain Reaction methods
- Abstract
Objectives: HIV drug resistance is a major concern as the emergence of resistant strains of virus results in failure of first-line therapies with an associated increase in the cost of subsequent regimens. Genotypic resistance is currently assessed by direct sequencing and cannot detect resistant species below 20%. Real-time PCR amplification was assessed for its ability to detect the signature mutation for nelfinavir, D30N., Methods: A real-time PCR assay was optimized for detection of low levels of D30N and tested on in vitro-generated nelfinavir-resistant isolates as well as 10 clinical isolates (which were also characterized by sequencing)., Results: The sensitivity of the assay was 1% and quantification was possible as low as 4% of the total viral population. Furthermore, this methodology enabled quantification of the 30N mutation in two isolates shown to be negative by sequencing., Conclusions: Real-time PCR is a promising tool for the detection of minority species of HIV but further studies are required to determine the specificity of the assay in a larger and thus more diverse set of clinical isolates.
- Published
- 2007
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