Crispr/Cas12a-Based Magnetic Relaxation Switching Biosensor for Nucleic Acid Amplification-Free and Ultrasensitive Detection of Methicillin-Resistant Staphylococcus Aureus

Methicillin-resistant Staphylococcus aureus (MRSA), as a superbug, has caused great harm to human health through the food chain. Current methods for MRSA detection primarily rely on the polymerase chain reaction, but require trained technicians and are easily false-positive. Herein, we develop a CRISPR/Cas12a-based magnetic relaxation switching (C-MRS) biosensor for ultrasensitive and nucleic acid amplification-free detection of MRSA in food. By incorporating collateral activity of CRISPR/Cas12a, on-particle rolling circle amplification, and alkaline phosphatase-triggered click chemistry into background-free MRS, as low as 16 CFU/mL MRSA can be detected without any nucleic acid pre-amplification, which avoids carryover contamination, but without compromising sensitivity. Moreover, this C-MRS biosensor could distinguish 0.01% target DNA from the single-base mutant. Recovery in eggs, milk and pork ranged from 75% to 112%, 82% to 104%, and 81% to 91%, respectively, revealing its satisfactory accuracy and applicability in the complex food matrix. The developed C-MRS biosensor fleshes out the CRISPR toolbox for food safety and provides a new approach for the sensitive and accurate detection of foodborne drug-resistant bacteria.