A Cycling Probe Technology (CPT) assay with a lateral-flow device (strip) was developed for the detection of the mecA gene from methicillin-resistant Staphylococcus aureus (MRSA) cultures. The assay uses a mecA probe (DNA-RNA-DNA) labeled with fluorescein at the 5' terminus and biotin at the 3' terminus. The CPT reaction occurs at a constant temperature, which allows the probe to anneal to the target DNA. RNase H cuts the RNA portion of the probe, allowing the cleaved fragments to dissociate from the target DNA, making the target available for further cycling. The strip detection step uses a nitrocellulose membrane with streptavidin and immunoglobulin G antibody impregnated on the surface. In the absence of the mecA gene, the uncut probe is bound to an antifluorescein-gold conjugate and is then captured by the streptavidin to form a test line. In the presence of the mecA gene, the probe is cut and no test line is formed on the strip. A screen of 324 S. aureus clinical isolates by the CPT-strip assay showed a 99.4% sensitivity and a 100% specificity compared to the results of PCR for the detection of the mecA gene. Specificity testing showed that the CPT-strip assay did not exhibit any cross-reactivity with a panel of mecA-negative non-S. aureus isolates. The CPT-strip assay is simple and does not require sophisticated equipment. Furthermore, the assay takes 1.5 h starting from a primary culture to the time to detection of the mecA gene in S. aureus isolates.