Multiple signal amplification strategy for ultrasensitive sensing of Mycobacterium bovis based on 8-17 DNAzyme and CRISPR-Cas13a.

Bovine tuberculosis caused by Mycobacterium bovis is not only responsible for economic losses but can also seriously jeopardize human health. Therefore, for the ultra-sensitive detection of M. bovis, a novel triple-cycle amplification system was developed based on 8-17 deoxyribozyme (DNAzyme), clustered regularly interspaced short palindromic repeats-associated protein 13a (CRISPR-Cas13a)-mediated cleavage cycles and the catalytic hairpin assembly (CHA) reaction (termed as DzCCR). In the presence of the target, the A-sequence containing 8-17 DNAzyme fragments was released from A-B using a strand displacement reaction, which could specifically cleave the HX-gRNA probe, releasing the sequence of gRNA and H and realizing the first signal amplification. Then, the released gRNA could bind to the Cas13a-g complex and activate the trans-cutting ability of Cas13a to re-cut RNA bulge sequences in HX-gRNAs, achieving the second signal amplification. Moreover, the H-sequence generated by the upstream 8-17 DNAzyme and Cas13a cleavage reaction further triggered the CHA, allowing the G-quadruplex dimer to be exposed, realizing signal output by adding thioflavin T (THT), and thereby achieving the third signal amplification. Benefiting from the triple signal amplification, the DzCCR system could quantitatively detect the M. bovis target down to a concentration of 0.5 fM with a linear calibration range from 1 to 500 fM. Furthermore, we investigated the ability of this system to detect M. bovis in real samples by standard addition method, the recovery ranged from 92.6% to 107.5%, and the relative standard deviations (RSD) ranged from 1.9% to 4.1%. Owing to the constant temperature and high sensitivity, the proposed strategy could be used as a new approach for the detection of M. bovis.