A programmable CRISPR/Cas12a-mediated hyperbranched hybridization chain reaction for ultrasensitive sensing of Mycobacterium bovis

Mycobacterium bovis results in a serious threat to domestic animal species, wildlife, and humans. Therefore, a novel colorimetric sensor with high sensitivity and specificity was developed for diagnosing Mycobacterium bovis by combining CRISPR/Cas12a with hyperbranched hybridization chain reaction (HB-HCR). In the presence of target DNA, the active Cas12a protein endowed with trans-cleavage activity showed its role in scgRNA. Thus, an auto-catalytic circuit was formed after the released gRNA molecules were converted to form additional active Cas12a effectors. Simultaneously, the released RNA initiator was used to trigger a cascade of HB-HCR events integrated G-quadruplex. Further, the rather low abundance of target could be observed with naked eyes when Hemin, H2O2 and ABTS were added. The results revealed that the limit of detecting Mycobacterium bovis target was 8.9 aM with single-nucleotide mismatch discrimination. Furthermore, the practical application of our designed colorimetric sensor for detecting Mycobacterium bovis performed by the standard addition method, the recovery ranged from 95.7% and 108.9% and the relative standard deviations (RSD) ranged from 1.2% and 2.8%, opened up a new avenue for highly sensitive and specific diagnosis of Mycobacterium bovis in real biological samples.