One-tube RPA-CRISPR Cas12a/Cas13a rapid detection of methicillin-resistant Staphylococcus aureus

At present, methicillin-resistant Staphylococcus aureus (MRSA) has caused a serious impact on a global scale. The infection and carrier rate of MRSA in the community is increasing year by year, but there is still no convenient detection system for on-site rapid detection. It is very important to select a rapid detection system to accurately and quickly detect patients infected with MRSA. We have developed a high-efficient single-tube detection platform based on RPA and CRISPR reaction system to detect the genes of mecA and clfA of MRSA. Using this detection platform, visual MRSA detection could be achieved in 30 min. It was observed that this detection platform was capable to successfully detect the target genomic as low as 5 copies μL−1, and the reaction was completed in one step without opening the lid. This detection platform could only detect MRSA, but not other common clinical pathogenic bacteria, such as Salmonella, Pseudomonas aeruginosa, Staphylococcus xylosus, Aeromonas hydrophila, Escherichia coli and Staphylococcus warneri, indicated its satisfactory selectivity for MRSA without interference from other bacteria. The results of clinical samples show that the platform has outstanding advantages in sensitivity, specificity and identification of methicillin resistance. The entire reaction can be completed in one step in the handheld instrument without opening the cover, avoiding aerosol pollution during the reaction. The detection platform combined with handheld instruments will have great application potential in point-of-care testing. The multiplexed RPA method is used to amplify two targets. The mecA DNA amplicons are identified directly by Cas12a-crRNA complexes, while the clfA DNA amplicons are transcribed into RNA using T7 transcription and detected by Cas13a-crRNA complexes. Orthogonal cleavage of DNA and RNA reporters by target activated Cas12a/Cas13a induces two colored fluorescence emission. [Display omitted] •The rapid, constant temperature reaction of this detection system is more suitable for POCT.•The whole reaction is completed in one step, reducing aerosol contamination.•The amplification of RPA combined with the CRISPR detection results in a more accurate and sensitive detection system.•The system could detect dual genes of Staphylococcus aureus and identify whether the strain is methicillin-resistant.