基于双功能杂交链式反应与CRISPR/Cas12a串联式启动酶促信号放大的牛结核分枝杆菌高灵敏比色传感研究OA

Mycobacterium bovis is a Gram-positive bacterium that affects livestock health and poses a public health risk.Rapid and accurate detection of low-abundance Mycobacterium bovis DNA in bovine blood is crucial for early diagnosis,disease control,and livestock management.In this study,a highly sensitive"turn-off"colorimetric sensing technique was developed for detecting Mycobacterium bovis based on dual-function hybridization chain reaction(HCR)coupled with clustered regularly interspaced short palindromic repeats(CRISPR)-CRISPR associated proteins 12a(CRISPR/Cas12a)enzyme-cascading signal amplification.In the presence of Mycobacterium bovis DNA targets,the DNA hairpins H1 and H2 were triggered to undergo HCR,forming DNA nanowire-like structures with multiple protospacer adjacent motif(PAM)sites.These structures were recognized by Cas12a/gRNA complexes,activating both cis-and trans-cleavage activities ofCRISPR/Cas12a.In the single-chain guide RNA(scgRNA),the iDNA-F and iDNA-B probes were designed with mismatched base sequences to construct a bulged structure,serving as a substrate for Cas12a's trans-cleavage activity.Upon cleavage,the double-stranded structure destabilized due to reduced Tm values,releasing the inhibitor probe embedded in the scgRNA.The released inhibitor probe bound to the downstream HP1 probe,effectively preventing the spontaneous assembly of HP1 and HP2 into G-quadruplex structures and subsequently blocking the formation of G-quadruplex-hemin DNAzyme.This resulted in a significant reduction in absorption signals.Under optimal experimental conditions,this method exhibited a linear detection range for Mycobacterium bovis-specific sequences from 1 nmol/L to 100 nmol/L,with a detection limit of 76 pmol/L(LOD=3σ/S).The regression equation was ΔA420 nm=0.09956 lgC+0.0555(R2=0.9845).When applied to detection of Mycobacterium bovisin bovine blood,the spiked recoveries were 98.3%to 108.0%.This technique was simple,highly selective,and sensitive,making it suitable for rapid and sensitive detection of Mycobacterium bovis in bovine blood.