A CRISPR/Cas13a-based detection method for the drug resistance gene mecA of Staphylococcus aureus

Abstract:

[Objective] To address the problem that the detection of drug resistance gene usually relies on specialized devices and the lack of rapid detection methods, we aimed to establish a rapid detection method for the drug resistance gene mecA of Staphylococcus aureus based on clustered regularly interspaced short palindromic repeats (CRISPR). [Methods] Firstly, we designed and screened the recombinase-aided amplification (RAA) primers and CRISPR RNA (crRNA) with high sensitivity according to the conserved region of mecA. Then, we established the detection method for mecA with the easy-readout and sensitive enhanced (ERASE) strip. Finally, we used simulated samples and clinical isolated samples to compare the established method with the traditional method. [Results] A set of efficient RAA primers and crRNAs targeting mecA were successfully screened out, and a highly sensitive CRISPR-ERASE-based nucleic acid detection method was established for mecA. This method can detect mecA at a minimum concentration of 10 copies/μL. Among 32 clinical isolates of S. aureus, 24 strains were tested positive for mecA by the established method, which was in 100% agreement with the results of antimicrobial susceptibility test and quantitative real-time PCR (qPCR). [Conclusion] A CRISPR-ERASE-based simple and sensitive method for detecting the drug resistance gene mecA was established.

Key words:CRISPR;isothermal amplification;mecA;nucleic acid detection;ERASE strips