Provided are methods of detecting the presence and/or amount of methicillin-resistant S. aureus in a sample. The methods may include, in some embodiments, processing the sample with a lysyl endopeptidase followed by amplification and detection of S. aureus chromosomal DNA if present in the sample. The PCR amplification may include the use of one or more forward primers that target SCCmec right chromosomal junction regions, a reverse primer that targets an orfX region and at least one probe that targets and orfX region between the forward and reverse primers. The reverse primer and/or the at least one probe may be targeted to conserved regions of orfX, i.e. regions that do not contain SNPs or other polymorphisms. Sets of oligonucleotides and kits for performing the methods are also provided.