title: Simplified protocol for pulsed-field gel electrophoresis analysis of Streptococcus pneumoniae.
abstract: A variety of pulsed-field gel electrophoresis (PFGE) protocols for the molecular subtyping of Streptococcus pneumoniae have been reported; most are time-consuming and complex. We sought to modify reference PFGE protocols to reduce the time required while creating high-quality gels. Only protocol modifications that resulted in high-quality banding patterns were considered. The following protocol components were modified. Lysis enzymes (lysozyme, mutanolysin, and RNase A) were deleted in a stepwise fashion, and then the lysis buffer was deleted. Lysis and digestion were accomplished in a single step with EDTA and N-lauroyl sarcosine (ES; pH 8.5 to 9.3) incubation at 50 degrees C in the absence of proteinase K. All enzymes except the restriction enzyme were omitted. A minimum incubation time of 6 h was required to achieve high-quality gels. All of the reactions were performed within 9 h, and the total protocol time from lysis to gel completion was reduced from 3 days to only 36 h. Combining lysis and digestion into a single step resulted in a substantial reduction in the time required to perform PFGE for S. pneumoniae. The ES solution may have caused cell lysis by activating N-acetylmuramyl-L-alanine amidase, the pneumococcal autolysin.
authors.affiliation: Public Health Infectious Diseases Laboratory, University of Pittsburgh Graduate School of Public Health, Pittsburgh, Pennsylvania, USA. McEllistremC@msx.dept-med.pitt.edu
authors.forename: M C
authors.initials: MC
authors.lastname: McEllistrem
authors.forename: J E
authors.initials: JE
authors.lastname: Stout
authors.forename: L H
authors.initials: LH
authors.lastname: Harrison
mesh_terms: D015373:Bacterial Typing Techniques; D001433:Bacteriolysis; D004492:Edetic Acid / Q000494:pharmacology; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*; D004789:Enzyme Activation; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000378:metabolism; D012150:Polymorphism, Restriction Fragment Length; D012521:Sarcosine / Q000031:analogs & derivatives / Q000494:pharmacology; D013296:Streptococcus pneumoniae / Q000145:classification / Q000235:genetics*
chemical_list: C025231:sarkosyl; D004492:Edetic Acid; D009238:N-Acetylmuramoyl-L-alanine Amidase; D012521:Sarcosine