Aptamer-based colorimetric detection of methicillin-resistant Staphylococcus aureus by using a CRISPR/Cas12a system...

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.