Rapid and sensitive detection of methicillin-resistant Staphylococcus aureus is crucial for effective treatment and control of clinical infections caused by this bacterium. The goal of this study is to develop a CE-based detection method for multiplexed identification of a femA sequence specific for S. aureus and a unique mecA sequence encoding methicillin resistance. Blood samples spiked with known concentrations of bacteria were used for testing. Crude cell lysates were prepared by treating the spiked blood samples with DNazol Direct reagent and used as the template for isothermal amplification of mecA and femA genes. The amplified gene products then underwent a cycling probe reaction (CPR)-based assay to generate a short fluorophore-labeled oligonucleotide for detection in a CZE-LIF system. The assay enables a gene-specific fluorophore-labeled DNA-RNA-DNA chimeric probe to hybridize with complementary target in the presence of RNase H enzyme. The RNase H enzyme specifically cleaves probe RNA residues of the duplex, releasing a fluorophore fragment for detection and the target for recycling and hybridization with another chimeric probe. Intact and cleaved probe fragments were separated and detected using a CZE-LIF system. The limit of detection for isothermal amplification and CPR-CZE-LIF was similar to 10(4) colony-forming units of bacteria/ml, of blood. This method accurately detects methicillin-resistant S. aureus within 3 h.