Colorimetric and fluorescent dual-identification of Methicillin-Resistant Staphylococcus aureus (MRSA) using programmable CRISPR/Cas12a system

Accurate identification of Staphylococcus aureus (S. aureus) is crucial for food safety and clinical diagnosis. Particularly, identifying drug-resistant strains guides effective treatment and prevention of infectious diseases. Herein, we presented a highly efficient programmable CRISPR/Cas12a system for identification of methicillin-resistant S. aureus (MRSA). Genus-specific gene (spec gene) and methicillin-resistant gene (mecA gene) of S. aureus were amplified by PCR. According to the sequences of spec gene and mecA gene, corresponding pro-grammable crRNAs were designed and prepared to construct CRISPR/Cas12a system, which recognized and bound to the PCR products through complementary interactions to activate the trans-cleavage activity of Cas12a. This led to the indiscriminate digestion of surrounding FQ-reporter, releasing a distinct fluorescence signal for MRSA identification. In addition, we devised a pair of AuNPs-DNA probes for colorimetric recognition of S. aureus. Target-triggered trans-cleavage for linker probe led to an non-aggregation of AuNPs-DNA probes pair. This led to a noticeable appearance of red color, enabling naked-eye detection. Thus, MRSA identification was realized with remarkable sensitivity and exceptional selectivity. Successful identification of MRSA in clinical samples validated the practical applications of the proposed CRISPR/Cas12a assay, establishing it as a universal platform for diagnosing pathogenic microorganisms with versatility and compatibility.