Rapid identification of methicillin-resistant Staphylococcus aureus and simultaneous species confirmation using real-time fluorescence PCR

Abstract

A duplex LightCycler PCR assay targeting the mecA gene and a Staphylococcus aureus-specific marker was used to test 165 S. aureus strains and 80 strains of other bacterial species. Within an assay time of 60 min plus 10 min for sample preparation, S. aureus as well as the presence or absence of the mecA gene was correctly identified.

FIG. 1

Analytical sensitivity of the PCR assay for the mecA gene (A) and the S. aureus-specific genomic fragment Sa442 (B), determined with serial dilutions of a cultured MRSA strain. Detection limits in terms of numbers of CFU, determined by a standard plating procedure, are given next to the corresponding amplicon curves.

FIG. 2

Evaluation of the PCR assay with clinical samples. A representative set of clinical samples was simultaneously tested for the mecA gene (A) and the S. aureus-specific marker (B). Amplicon curves representing mecA-positive (pos.) isolates are indicated by brackets. The mecA-negative (neg.) samples of this panel were two MSSA strains. The S. aureus-negative sample in this panel was a methicillin-resistant strain of S. epidermidis.