Technical aspects of random amplified polymorphic DNA (RAPD) technique in genotyping of bacterial strains.
Random Amplified Polymorphic DNA (RAPD) method became widely applied for sensitive, efficient and fast distinguishing of different isolates of a given species, if pure culture is available. Problems with reproducibility and discriminatory power, frequently cited in the literature, can be overcome by precise optimization procedure allowing to achieve reliable conditions for each species analysed. Basing on two examples of different species, H. pylori and E. faecium, particular parameters of RAPD fingerprinting were evaluated with respect to selection of best working primers generating medium-complex profiles, using only high quality DNA samples and evaluation optimum for every reaction reagent. Stable and informative amplification patterns were obtained with different best working primers which could discriminate between all H. pylori and E. faecium strains tested. For both analysed species different optima were found, suggesting species-specific need of precise RAPD conditions evaluation. This study proved high sensitivity and efficiency of optimized RAPD profiling applicable for searching the epidemiology traces for both species.
Gzyl
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Department of Clinical Microbiology, Children's Memorial Health Institute, Warsaw, Poland.
Augustynowicz
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D015373:Bacterial Typing Techniques; D004269:DNA, Bacterial / Q000032:analysis; D016984:Enterococcus faecium / Q000145:classification* / Q000235:genetics*; D005838:Genotype; D016908:Gram-Positive Bacterial Infections / Q000382:microbiology; D016481:Helicobacter Infections / Q000382:microbiology; D016480:Helicobacter pylori / Q000145:classification* / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D019105:Random Amplified Polymorphic DNA Technique; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial