Multiplexed genotyping of methicillin-resistant Staphylococcus aureus isolates by use of padlock probes and tag...

We developed and tested a ligase-based assay for simultaneous probing of core genome diversity and typing of methicillin resistance determinants in Staphylococcus aureus isolates. This assay uses oligonucleotide padlock probes whose two ends are joined through ligation when they hybridize to matching target DNA. Circularized probes are subsequently amplified by PCR with common primers and analyzed by using a microarray equipped with universal tag probes. Our set of padlock probes includes oligonucleotides targeting diagnostic regions in the mecA, ccrB, and ccrC genes of the SCCmec cassette in methicillin-resistant S. aureus (MRSA). These probes determine the presence and type of SCCmec cassettes (i.e., SCCmec types I to VI). Additional oligonucleotides interrogate a number of highly informative single nucleotide polymorphisms retrieved from a multilocus sequence typing (MLST) database. These latter probes enable the exploration of isolates' phylogenetic affiliation with clonal lineages of MRSA as revealed by MLST. The described assay enables multiplexed genotyping of MRSA based on a single-tube reaction. With a set of clinical isolates of MRSA and methicillin-susceptible S. aureus (n=66), 100% typeability and 100% accuracy were achieved. The assay described here provides valuable genotypic information that may usefully complement existing genotyping procedures. Moreover, the assay is easily extendable by incorporating additional padlock probes and will be valuable for the quick and cost-effective probing of large numbers of polymorphisms at different genomic locations, such as those ascertained through currently ongoing mutation discovery and genome resequencing projects.