The construction of CRISPR/Cas9-mediated FRET 16S rDNA sensor for detection of Mycobacterium tuberculosis .

The simple and efficient detection of nucleic acids is important in the diagnosis of tuberculosis (TB) caused by Mycobacterium tuberculosis ( M. tuberculosis ). However, base mismatch will lead to false positive and false negative nucleic acid test, which seriously interferes with the accuracy of the final results. Herein, we demonstrated a CRISPR/Cas-9-mediated fluorescent strategy utilizing fluorescence resonance energy transfer (FRET) for the detection of bacteria. High-variable region of M. tuberculosis 16S rDNA fragment was used as the target, and CRISPR/Cas9 was used as the recognition element. The binding of the P1 probe of upconversion nanoparticles (UCNPs) @SiO 2 -P1 and the P2 probe of Fe 3 O 4 @Au-P2 caused the fluorescence quenching of UCNPs. In the presence of the target, the P2 probe hybridized with the target to form double-stranded DNA (dsDNA), which was recognized and cleaved by CRISPR/Cas9, resulting in the breaking of the P1-P2 duplex linkage. UCNPs moved away from Fe 3 O 4 @Au under a magnetic field, and the fluorescence signal was restored; bacteria were detected under the excitation of a 980 nm laser source. Using the CRISPR/Cas-9-mediated system, the sensor could distinguish single-base mismatches in 10 bases from the protospacer adjacent motif (PAM) region. The limit of detection (LOD) was 20 CFU mL -1 and the detection time was 2 h. It developed a new way of accurate nucleic acid detection for disease diagnosis.