Microfluidic Padlock Probe-based Rolling Circle Amplification for sensitive detection of mecA resistance gene in Staphylococcus aureus

Antimicrobial resistance and the dissemination of resistance genes remain critical global health challenges, demanding efficient monitoring and detection tools. Methicillin-resistant Staphylococcus aureus (MRSA) is a prominent example, posing serious clinical and epidemiological concerns. Here, we developed a sensitive detection platform combining isothermal Padlock Probe-based Rolling Circle Amplification with a bead-based microfluidic biosensor and fluorescent readout for the detection of the mecA gene in S. aureus . The assay design was first validated using a complementary synthetic ssDNA target, then optimized with genomic dsDNA extracted from a laboratory strain. The optimized system was subsequently applied to clinical isolates from sepsis cases and complex matrices simulating natural habitats, including milk, serum, and salad. The biosensor achieved a detection limit of 10 4 cells with 100 % specificity compared to PCR at an analysis time of two hour. All reaction steps were carried out sequentially within the microfluidic device, highlighting the strong potential of the device for full automation and for extension to additional bacterial genes. • Padlock Probe-based Rolling Circle Amplification fluorescent microfluidic gene detection technology. • Screening of methicillin-resistant Staphylococcus aureus based on mecA gene detection. • mecA gene detection in S. aureus clinical isolates related to sepsis. • The direct detection of mecA in bacterial lysates from green salad wash water. • The efficient detection of mecA in lysates of washed bacterial cells from milk.