Simplified Protocol for Pulsed-Field Gel Electrophoresis Analysis of Streptococcus pneumoniae

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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°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.