Aptamer-based colorimetric detection of methicillin-resistant Staphylococcus aureus by using a CRISPR/Cas12a system and recombinase polymerase amplification br

Detection of methicillin-resistant Staphylococcus aureus (MRSA) with superior accuracy, timeliness, and simplicity is highly valuable in clinical diagnosis and food safety. In this study, an aptamer-based colorimetric biosensor was developed to detect MRSA by using a CRISPR/Cas12a system and recombinase polymerase amplification (RPA). The aptamer of silver ion (Ag+) pre-coupled to magnetic nanoparticles was employed not only as the substrate of trans-cleavage in the CRISPR/Cas12a system, but also as the modulator of Ag+-3,3 ',5,5 ' tetramethylbenzidine (TMB) chromogenic reaction, innovatively integrating the powerful CRISPR/Cas12a system with convenient colorimetry. The utilized aptamer containing consecutive and interrupted cytosine: cytosine mismatched base pairs also served as a signal amplifier because of the one-to-multiple binding of the aptamer to Ag+. Using triple amplification of RPA, multiple-turnover nuclease activity of Cas12a, and cytosine-Ag+-cytosine coordination chemistry, MRSA was detected as low as 8 CFU mL-1. Moreover, its satisfactory accuracy in the analysis of real samples, together with visualization and simplicity, revealed the great potential of the proposed biosensor as a robust antibiotic-resistant bacteria detection platform.

Automated summary

Aptamer-based colorimetric biosensor using CRISPR/Cas12a and RPA was developed for sensitive and convenient detection of MRSA. The biosensor integrated the CRISPR/Cas12a system with colorimetry by employing an aptamer that served as the substrate and modulator. Multiple amplification strategies and signal amplification were employed to enhance the sensitivity, and the biosensor exhibited high accuracy and simplicity in detecting MRSA in real samples, suggesting its potential as a robust antibiotic-resistant bacteria detection platform.