Simplified protocol for pulsed-field gel electrophoresis analysis of Streptococcus pneumoniae
M C McEllistrem1, J E Stout, L H Harrison
1Public Health Infectious Diseases Laboratory, University of Pittsburgh Graduate School of Public Health, Pittsburgh, Pennsylvania, USA. McEllistremC@msx.dept-med.pitt.edu
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.
Insights
This study optimized pulsed-field gel electrophoresis (PFGE) for Streptococcus pneumoniae subtyping, significantly reducing protocol time from 3 days to 36 hours while maintaining gel quality. The new method combines lysis and digestion steps for faster molecular subtyping.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Pulsed-field gel electrophoresis (PFGE) is crucial for molecular subtyping of bacterial pathogens like Streptococcus pneumoniae.
- Existing PFGE protocols for S. pneumoniae are often lengthy and complex, hindering rapid diagnostics and research.
Purpose of the Study:
- To modify and streamline existing PFGE protocols for S. pneumoniae.
- To reduce the overall time required for PFGE while ensuring high-quality results.
Main Methods:
- Stepwise deletion of lysis enzymes (lysozyme, mutanolysin, RNase A) and lysis buffer.
- Combined lysis and digestion in a single step using EDTA and N-lauroyl sarcosine (ES) buffer at 50°C, omitting proteinase K.
- Optimization of incubation times, requiring a minimum of 6 hours for high-quality gels.
Main Results:
- A simplified PFGE protocol was developed, reducing the total procedure time from 3 days to 36 hours.
- High-quality banding patterns were consistently achieved with the modified protocol.
- The ES solution potentially facilitates cell lysis by activating pneumococcal autolysin.
Conclusions:
- The optimized PFGE protocol offers a significantly faster method for S. pneumoniae molecular subtyping.
- This streamlined approach can improve the efficiency of epidemiological surveillance and research involving S. pneumoniae.
- The simplified protocol maintains the integrity and quality of PFGE results.
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