New real-time PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus directly from specimens containing a mixture of staphylococci

Abstract

Molecular methods for the rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) are generally based on the detection of an S. aureus-specific gene target and the mecA gene. However, such methods cannot be applied for the direct detection of MRSA from nonsterile specimens such as nasal samples without the previous isolation, capture, or enrichment of MRSA because these samples often contain both coagulase-negative staphylococci (CoNS) and S. aureus, either of which can carry mecA. In this study, we describe a real-time multiplex PCR assay which allows the detection of MRSA directly from clinical specimens containing a mixture of staphylococci in <1 h. Five primers specific to the different staphylococcal cassette chromosome mec (SCCmec) right extremity sequences, including three new sequences, were used in combination with a primer and three molecular beacon probes specific to the S. aureus chromosomal orfX gene sequences located to the right of the SCCmec integration site. Of the 1,657 MRSA isolates tested, 1,636 (98.7%) were detected with the PCR assay, whereas 26 of 569 (4.6%) methicillin-susceptible S. aureus (MSSA) strains were misidentified as MRSA. None of the 62 nonstaphylococcal bacterial species or the 212 methicillin-resistant or 74 methicillin-susceptible CoNS strains (MRCoNS and MSCoNS, respectively) were detected by the assay. The amplification of MRSA was not inhibited in the presence of high copy numbers of MSSA, MRCoNS, or MSCoNS. The analytical sensitivity of the PCR assay, as evaluated with MRSA-negative nasal specimens containing a mixture of MSSA, MRCoNS, and MSCoNS spiked with MRSA, was approximately 25 CFU per nasal sample. This real-time PCR assay represents a rapid and powerful method which can be used for the detection of MRSA directly from specimens containing a mixture of staphylococci.

FIG. 1.

Schematic representation of the MREJs of MRSA strains with MREJ types i to vii. Each MREJ type exhibits a distinct SRE sequence, with the exception of MREJ types i and ii, which differ by a 102-bp insertion. The positions of forward (▸) and reverse (◂) primers and of internal probes (bars) included in the MRSA assay are shown. Three MBPs, XsauB5, XsauB8, and XsauB9, were used to detect the MRSA-specific amplification products. The locations within SCCmec of the crr complex and the mec complex containing mecA are shown, but these genetic elements are not drawn to scale. The vertical arrow at the top indicates the SCCmec integration site within the chromosomal S. aureus orfX gene. The half- arrows show the positions of the orfX start codon and the direction of transcription. More details regarding primers and probes are found in Table 3.

FIG. 2.

Example showing the FAM fluorescence detection of MRSA, using 10 copies of genomic DNAs purified from MRSA strains with MREJ types i (solid line), ii (dashed line with dots), iii (circles), iv (solid line with circles), v (solid line with hash marks), and vii (squares). Dashed line, negative control.