| Autor: |
Ivanov AV; A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia., Safenkova IV; A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia., Zherdev AV; A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia., Wan Y; State Key Laboratory of Marine Resource Utilization in South China Sea, Marine College, Hainan University, Haikou 570228, China., Dzantiev BB; A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia. |
| Abstrakt: |
Biosensors based on endonuclease Cas12 provide high specificity in pathogen detection. Sensitive detection using Cas12-based assays can be achieved using trans-cleaved DNA probes attached to simply separated carriers, such as magnetic particles (MPs). The aim of this work was to compare polyA, polyC, and polyT single-stranded (ss) DNA with different lengths (from 10 to 145 nt) as trans-target probes were immobilized on streptavidin-covered MPs. Each ssDNA probe was labeled using fluorescein (5') and biotin (3'). To compare the probes, we used guide RNAs that were programmed for the recognition of two bacterial pathogens: Dickeya solani (causing blackleg and soft rot) and Erwinia amylovora (causing fire blight). The Cas12 was activated by targeting double-stranded DNA fragments of D. solani or E. amylovora and cleaved the MP-ssDNA conjugates. The considered probes demonstrated basically different dependencies in terms of cleavage efficiency. PolyC was the most effective probe when compared to polyA or polyT probes of the same length. The minimal acceptable length for the cleavage follows the row: polyC < polyT < polyA. The efficiencies of polyC and polyT probes with optimal length were proven for the DNA targets' detection of D. solani and E. amylovora . The regularities found can be used in Cas12a-based detection of viruses, bacteria, and other DNA/RNA-containing analytes. |
