A novel one-tube RPA/CRISPR melting curve detection sensing system based on unique 3'-toehold nucleic acid aptamer...

Developing effective detection methods for Bacillus anthracis is essential for our public health system to accurately detect hidden anthrax outbreaks. Herein, we introduce a unique 3'-toehold nucleic acid aptamer (probes) into Cas12a biosensor, combined with RPA, to establish a rapid (1h), specific, and sensitive (1copy/μL) detection method for B. anthracis. The design behind this approach is that the target sequence is amplified via RPA, and the amplification product triggers the crRNA/Cas12a complex to degrade the 3' toehold probes, which are analyzed using melting curve analysis on a specific instrument, naming a one-tube RPA/CRISPR melting curve detection (ORCMD) sensing system. Furthermore, ORCMD is used to detect the B. anthracis spores-positive or negative soil samples from the location of world War-II site (Harbin, China), B. anthracis was precisely identified as other methods, suggesting its significant practical application potential. This system enriches the CRISPR detection technology toolbox, compared to other CRISPR-based sensing strategies, the concept of the 3' toehold probes offers distinct advantages in the development of CRISPR-based multi-target detection methods.