Development of an optimized random amplified polymorphic DNA protocol for fingerprinting of Klebsiella pneumoniae

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 1μmol1^-1 primer, 4mmol1^-1 MgCl₂, 0·4mmol1^-1 dNTPs, 2·5UTaq DNA polymerase and 90ng DNA template in a total volume of 25μl. The optimum thermocycling program was: initial denaturation at 94°C for 4min followed by 50 cycles of 1min at 94°C, 2min at 34°C, 2min at 72°C and a final extension at 72°C for 10min. 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.