Rapid multiplex PCR assay for identification of USA300 community-associated methicillin-resistant Staphylococcus...

Area of Science:

• Microbiology
• Molecular Biology
• Infectious Diseases

Background:

• Increasing incidence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections in individuals lacking traditional risk factors.
• The prevalent CA-MRSA strain in the US, USA300, is characterized by Panton-Valentine leukocidin (PVL) genes and staphylococcal cassette chromosome mec (SCCmec) type IV.
• Current molecular identification methods for MRSA strains like USA300 are often slow and technically demanding for clinical labs.

Purpose of the Study:

• To develop a rapid and accessible method for identifying the USA300 CA-MRSA strain.
• To investigate unique genetic markers associated with the USA300 strain for diagnostic purposes.

Main Methods:

• Analysis of chromosomal regions flanking the SCCmec element in 44 USA300 MRSA isolates.
• Identification of a signature "AT repeat" sequence in the SACOL0058 gene downstream of the SCCmec junction.
• Development of a multiplex PCR assay using locked nucleic acid primers for AT repeats and PVL detection.

Main Results:

• A specific "AT repeat" sequence was identified in close proximity to the SCCmec element in USA300 isolates.
• The presence of > or =6 AT repeats combined with PVL genes was unique to USA300 strains among those tested.
• The developed multiplex PCR assay demonstrated 100% concordance with DNA sequencing for USA300 identification.

Conclusions:

• The novel multiplex PCR assay provides a rapid and reliable method for identifying USA300 CA-MRSA strains.
• This assay can overcome the limitations of current time-consuming molecular characterization techniques.
• The findings support the utility of this assay for routine clinical laboratory use in identifying prevalent CA-MRSA strains.