RESUMO
AIMS: To develop an optimized random amplified polymorphic DNA (RAPD) protocol for fingerprinting clinical isolates of Klebsiella pneumoniae. METHODS AND RESULTS: Employing factorial design of experiments, repeatable amplification patterns were obtained for 54 nosocomial isolates using 1 µmol 1(-1) primer, 4 mmol 1(-1) MgCl(2), 0·4 mmol 1(-1) dNTPs, 2·5 U Taq DNA polymerase and 90 ng DNA template in a total volume of 25 µl. The optimum thermocycling program was: initial denaturation at 94°C for 4 min followed by 50 cycles of 1 min at 94°C, 2 min at 34°C, 2 min at 72°C and a final extension at 72°C for 10 min. The optimized RAPD protocol was highly discriminatory (Simpson's diversity index, 0·982), and all isolates were typable with repeatable patterns (Pearson's similarity coefficient ≈ 100%). Seven main clusters were obtained on a similarity level of 70% and 32 distinct clusters on a similarity level of 85%, reflecting the heterogeneity of the isolates. CONCLUSIONS: Systematic optimization of RAPD generated reliable DNA fingerprints for nosocomial isolates of K. pneumoniae. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on RAPD optimization based on factorial design of experiments for discrimination of K. pneumoniae.