Evaluation of an optimized system for random amplified polymorphic DNA (RAPD)-analysis for genotypic mapping of Candida albicans strains.
A simple, rapid, and cost-effective protocol has been developed for a PCR-based molecular typing method for Candida albicans, which includes the use of a commercially available medium (Chelex 100 Resin) for DNA extraction and a single set of two arbitrarily chosen oligonucleotide (10 nt length) primers for random amplified DNA(RAPD)-analysis. The optimized parameters for the amplification components and conditions for the selected primer combination have been determined to avoid artifactual variation (absence/presence of bands) in RAPD banding patterns in repeated assays. The optimized RAPD-assay consistently generated DNA-patterns of 33 genetically unrelated C. albicans isolates that contained ten polymorphic markers in the non-artifactual banding patterns. The intralaboratory reproducibility of RAPD patterns was efficient and consistent provided the optimized amplification conditions were rigidly controlled. Interlaboratory reproducibility was tempered by slight variations in time of cyclers of different thermocyclers. In comparison, the RAPD assay was almost equal to restriction enzyme analysis (REA) (Eco RI digested chromosomal DNA) in discrimination, and the RAPD assay was able to group isolates of C. albicans that were untypable by REA. The protocol outlined for an optimized RAPD-assay of C. albicans has the potential to be widely useful epidemiological screening tool that can be easily applied in the clinical laboratory.
Holmberg
K
K
Department of Microbiology, National University of Singapore.
Feroze
F
F
D002176:Candida albicans / Q000145:classification / Q000235:genetics*; D002411:Cation Exchange Resins; D002614:Chelating Agents; D002874:Chromosome Mapping / Q000379:methods* / Q000592:standards; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000032:analysis / Q000302:isolation & purification; D005069:Evaluation Studies as Topic; D005820:Genetic Testing; D005838:Genotype; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results; D012117:Resins, Synthetic
D002411:Cation Exchange Resins; D002614:Chelating Agents; D017931:DNA Primers; D004271:DNA, Fungal; D012117:Resins, Synthetic; C024997:Chelex 100