An isocaudomer-assisted AND-gate Cas12a biosensor for dual-gene identification of methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is commonly identified by detection of the mecA resistance gene, but reliance on a single marker can lead to false positives, since mecA also occurs in non-MRSA organisms. Here, we report an i̲socaudomer-assisted A̲N̲D̲-gate C̲a̲s̲12a biosensor (iCasAND) for improved discrimination of MRSA by detecting the S. aureus-specific nuc gene and the methicillin-resistance gene mecA. Amplicons of both genes are digested by isocaudomer restriction enzymes (BglII and BamHI) to generate complementary sticky ends that self-assemble into split dsDNA activators of Cas12a trans-cleavage. Fluorescence is produced only when both genes are present, satisfying the AND logic condition, while T4 DNA ligase promotes complete activator formation, enhancing the signal-to-background ratio. The iCasAND system achieves a detection limit of 7682.45 ag μL-1 and a sensitivity of 45.28 CFU mL-1, discriminates MRSA from closely related pathogens, and demonstrates 100% concordance in samples from an artificial tilapia infection model. This enzyme-assisted, logic-controlled Cas12a strategy enhances specificity without requiring multiplexed crRNAs and simplifies reagents and workflow, offering a highly specific, programmable, multi-step biosensing strategy for the detection of multidrug- resistant bacteria.