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Development of an optimized random amplified polymorphic DNA protocol for fingerprinting of Klebsiella pneumoniae
Author(s) -
Ashayeripanah M.,
Eftekhar F.,
Feizabadi M.M.
Publication year - 2012
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/j.1472-765x.2012.03203.x
Subject(s) - klebsiella pneumoniae , dna profiling , biology , microbiology and biotechnology , dna , genetics , enterobacteriaceae , escherichia coli , gene
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 .