MA L, KOVACS JA; Interscience Conference on Antimicrobial Agents and Chemotherapy.
Abstr Intersci Conf Antimicrob Agents Chemother Intersci Conf Antimicrob Agents Chemother. 2000 Sep 17-20; 40: 394.
NIH, Bethesda, MD
BACKGROUND: Recent studies have suggested that point mutations in human Pneumocystis carinii dihydropteroate synthase (DHPS) gene are involved in the development of resistance to sulfa drugs, which have been widely used for treatment and prophylaxis of P. carinii pneumonia (PCP). There is a clear need to develop a simple and rapid method for high-throughput screening of these mutations in a large-scale population.METHODS: Plasmids containing either wild-type or mutant human P. carinii DHPS sequences were used to optimize the assay conditions. Thirty-seven clinical P. carinii isolates of which the P. carinii DHPS sequences were determined by direct sequencing were used to evaluate the feasibility of single-strand conformation polymorphism (SSCP) analysis for mutation discrimination. A 259bp fragment spanning the mutation sites (codons 55 and 57) of the human P. carinii DHPS gene was amplified by PCR. The amplified products were denatured and electrophoresed in a small non-denaturing polyacrylamide gel. The bands were detected by silver staining.RESULTS: SSCP was able to detect unequal mixtures of pure wild-type genotype and single-base mutant genotype as long as the minority genotype constituted about 15% of the total. The 37 clinical isolates showed different SSCP patterns. Fifteen isolates containing either homozygous or heterozygous point mutations determined by direct DNA sequencing could be distinguished from 22 homozygous wild-type isolates by SSCP patterns. Four samples determined by direct DNA sequencing as homozygous wild-type or mutant genotypes showed heterozygous SSCP patterns. Conclusion: SSCP in combination with PCR is simple, relatively rapid, highly sensitive, and would serve as a screening method for detection of human P. carinii DHPS mutations.KEYWORDS: Dihydropteroate synthase; Pneumocystis carinii; Single-strand conformation polymorphism
Publication Types:
Keywords:
- Animals
- Dihydropteroate Synthase
- Genotype
- Humans
- Mutation
- Oxidoreductases Acting on CH-NH Group Donors
- Point Mutation
- Polymerase Chain Reaction
- Polymorphism, Single-Stranded Conformational
- deoxyhypusine synthase
- genetics
- methods
Other ID:
UI: 102248056
From Meeting Abstracts