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Related Experiment Videos

Denaturing gradient gel electrophoretic multilocus sequence typing of Staphylococcus aureus isolates.

Volker Gürtler1, Helen D Barrie, Barrie C Mayall

  • 1Department of Microbiology, Austin & Repatriation Medical Centre (Austin Campus), Heidelberg, Victoria, Australia. volker@austin.unimelb.edu.au

Electrophoresis
|October 10, 2002
PubMed
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A new denaturing gradient gel electrophoresis (DGGE) method offers a rapid, accurate, and cost-effective alternative to DNA sequencing for multilocus sequence typing (MLST) of Staphylococcus aureus. This approach enhances bacterial typing efficiency and reduces costs significantly.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • Multilocus sequence typing (MLST) is crucial for bacterial strain characterization.
  • Traditional MLST relies on DNA sequencing, which can be time-consuming and expensive.
  • Developing alternative methods for MLST is essential for broader applicability.

Purpose of the Study:

  • To develop a denaturing gradient gel electrophoresis (DGGE) based method for MLST of Staphylococcus aureus.
  • To assess the accuracy and cost-effectiveness of the DGGE-MLST method compared to traditional sequencing.
  • To establish optimized PCR and DGGE conditions for reliable strain typing.

Main Methods:

  • Polymerase chain reaction (PCR) amplification of seven housekeeping genes in Staphylococcus aureus isolates.

Related Experiment Videos

  • Optimization of PCR conditions with GC-clamps and selection of restriction enzyme sites for fragment digestion.
  • Analysis of PCR products using DGGE to generate DGGE types (DTs) and comparison with sequence types (STs).

    • Main Results:

    • The DGGE-MLST method accurately classified the majority of DTs (76/100) into single STs, detecting up to 95% of nucleotide changes.
    • A combined ST and DT approach allowed for the determination of septuplet sets of STs (7-ST).
    • The DGGE-MLST method proved to be 20-fold less expensive than DNA sequencing.

    Conclusions:

    • DGGE-MLST provides a rapid, accurate, and significantly more economical alternative to DNA sequencing for Staphylococcus aureus typing.
    • This method has potential for wide applicability in bacterial typing and screening multilocus nucleotide differences.
    • The combined laboratory and in silico approach offers a valuable tool for microbial genomics research.