CRISPR/Cas12a-based magnetic relaxation switching biosensor for nucleic acid amplification-free and ultrasensitive detection of methicillin-resistant Staphylococcus aureus

Abstract

Herein, we develop a CRISPR/Cas12a-based magnetic relaxation switching (C-MRS) biosensor for ultrasensitive and nucleic acid amplification-free detection of methicillin-resistant Staphylococcus aureus (MRSA) in food. In this biosensor, mecA gene in MRSA was recognized by CRISPR-RNA, which will activate the trans-cleavage activity of Cas12a and release the fastened alkaline phosphatase (ALP) on the particle. The freed ALP can then use to hydrolyze substrate to produce ascorbic acid that trigger the click reaction between magnetic probe. The transverse relaxation time of the unbound magnetic probe can be measured for signal readout. By incorporating collateral activity of CRISPR/Cas12a, on-particle rolling circle amplification, and ALP-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%–104%, and 81%–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.