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Updated: Aug 11, 2026
Detection of Bacteria Using Fluorogenic DNAzymes
Published on: May 28, 2012
Virtual digest identification of secondary enzymes for use in pulsed-field gel electrophoresis of Staphylococcus
Jill C Roberts1, Andrew C Cannons, Philip T Amuso
1Center for Biological Defense, University of South Florida College of Public Health, Tampa, FL 33612, USA. jroberts@bt.usf.edu
Abstract:
Pulsed-field gel electrophoresis (PFGE) is currently the gold standard for methicillin-resistant Staphylococcus aureus (MRSA) typing but only one enzyme, SmaI, is currently used for restriction digest. We report the use of virtual digestion to identify enzymes for S. aureus PFGE. Two enzymes (EagI and SacII) were identified and successfully used to characterize two sets of S. aureus isolates, 12 USA300, and 14 additional MRSA isolates comprised of seven SmaI patterns. Phylogenetic analysis of patterns generated by all enzymes determined that the USA300 MRSAs are identical. In contrast, digestion with EagI or SacII resolved one to two band differences among three MRSA pattern sets that were not detected using SmaI. These results demonstrate that a second enzyme may detect differences in S. aureus isolates not detected by single enzyme digestion. However, because isolates differing by one to two bands are considered identical, such discrimination may not be clinically or epidemiologically relevant.
Insights
This study explored new enzymes for pulsed-field gel electrophoresis (PFGE) to improve methicillin-resistant Staphylococcus aureus (MRSA) typing. Additional enzymes like EagI and SacII revealed subtle differences in MRSA strains not seen with SmaI alone.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Pulsed-field gel electrophoresis (PFGE) is the standard for typing methicillin-resistant Staphylococcus aureus (MRSA).
- Current MRSA typing relies on a single restriction enzyme, SmaI, potentially limiting discriminatory power.
Purpose of the Study:
- To identify alternative restriction enzymes for S. aureus PFGE using virtual digestion.
- To assess the utility of new enzymes in characterizing MRSA isolates and compare them with SmaI.
Main Methods:
- Virtual digestion was employed to identify candidate enzymes for S. aureus PFGE.
- Two enzymes, EagI and SacII, were selected and applied to characterize MRSA isolates.
- Phylogenetic analysis was performed on PFGE patterns generated by SmaI, EagI, and SacII.
Main Results:
- Virtual digestion identified EagI and SacII as suitable enzymes for S. aureus PFGE.
- All USA300 MRSA isolates were identical using SmaI, EagI, and SacII.
- EagI and SacII revealed one to two band differences in other MRSA sets, undetected by SmaI.
Conclusions:
- Employing additional enzymes like EagI or SacII can enhance the resolution of MRSA typing compared to SmaI alone.
- The clinical or epidemiological relevance of minor band differences detected by new enzymes requires further investigation.
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