Open AccessModified PFGE protocol for improving typeability of DNA degradation susceptible nosocomial Klebsiella pneumoniae
Author(s) -
Ahmad Zakaria,
Noha A. Hassuna
Publication year - 2019
Publication title -
journal of medical microbiology/journal of medical microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.91
H-Index - 117
eISSN - 1473-5644
pISSN - 0022-2615
DOI - 10.1099/jmm.0.001093
Subject(s) - klebsiella pneumoniae , pulsed field gel electrophoresis , microbiology and biotechnology , genomic dna , dna , biology , subtyping , gene , escherichia coli , biochemistry , genotype , computer science , programming language
. PFGE is the 'gold standard' method for bacterial subtyping. However, many strains are non-typable by this approach because of DNA degradation by nucleases action. Aim. To evaluate a modified PFGE protocol for typing nosocomial isolates of Klebsiella pneumoniae . Methods. Twenty- five K. pneumoniae isolates previously exposed to DNA degradation were used to optimize an extraction method for elimination of DNases activity before applying Xba1 enzyme. Introducing of sodium dodecyl sulfate (SDS) in different concentrations to the extraction buffer was evaluated for protecting genomic DNA molecule from degradation by nucleases. Results. Addition of 3 % SDS in combination with 3 % N-lauryl sarcosine to the extraction buffer was found to reduce the previously experienced nuclease activity. Pre-examination of plug quality prior to the digestion phase could efficiently reduce the expense of the wasted enzyme. Conclusion. We have successfully devised a PFGE protocol that enhanced the typeability of nosocomial K. pneumoniae .