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

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.