부산대학교 도서관

Objective: To compare a new point mutation assay with gene sequencing for the detection of the K103N and Y181C RT mutations. Methods: Plasma samples were obtained from patients failing HAART. These samples were sequenced (Visible Genetics) and were also tested using a novel 'in-house' assay for mutations at positions 103 and 181 of RT. The assay is an adaptation of 'Mutagenically Separated PCR' (MS-PCR, Rust et al.) and uses four primers in a three-round semi-nested reaction for each mutation. Results: Fifteen patient samples have been tested by both techniques. By sequencing, 4/15 samples were mutant (MT) for codon 103 and 10/15 were wild-type (WT). For codon 181, 6/15 patients were MT and 8/15 were WT. Twenty-four codons produced a result by both techniques, and of these 22/24 (91.7%) were identical. MS-PCR produced two MT codons which were not identified by sequencing. One patient sample failed to produce a result by sequencing and two failed by MS-PCR. Sensitivity of MSPCR for MT detection was 82%, with a specificity of 94.4%. Sequence data and gel electrophoresis images will be presented. Conclusions: MS-PCR is a new, cost-effective genotyping assay, which is simple and cheap to perform. There is no requirement for expensive hardware or software, lending the assay to high throughput screening. [ABSTRACT FROM AUTHOR]