The dCas9/crRNA linked immunological assay (dCLISA) for sensitive, accurate, and facile drug resistance gene analysis

The rapid and reliable diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) is essential for preventing the spread of MRSA infections and guiding therapeutic strategies. However, there is still a huge challenge in further simplifying MRSA detection procedures and improving detection selectivity to reduce false-positive results. In this study, we developed a derivative CRISPR-associated protein 9/CRISPR-derived RNA Linked Immunological Assay (dCLISA) for the sensitive and specific detection of MRSA. This technique utilizes two dCas9/crRNA complexes as specific targeting agents and employs a color reaction mediated by a hybridization chain reaction for signal output. The dCLISA method offers certain benefits compared to monoclonal antibodies in traditional immunoassays, primarily due to its capacity to selectively interact with target gene and its high sensitivity from the hybridization chain reaction process. Therefore, the minimum detectable concentration of dCLISA was 8.5 cfu/mL. Unlike traditional gene analysis approaches, target gene sequences in cell lysates can be directly detected by dCLISA within 60 min with high sensitivity without genomic material extraction. In addition, the absorbance intensity of the MRSA cell lysate was significantly higher than that of methicillin-susceptible S. aureus (MSSA) indicates the clinical application potential. This study demonstrates that the dCLISA is a simple, rapid, sensitive, and specific method, which can be directly used at the point of care to analyze drug resistance in bacteria, including MRSA. Moreover, dCLISA can be utilized for other bacteria detection by merely modifying the crRNA sequence.