Development of an MSPQC Nucleic Acid Sensor Based on CRISPR/Cas9 for the Detection of Mycobacterium tuberculosis

Accurate and rapid detection of nucleic acid plays a vital role in the clinical treatment of tuberculosis caused by Mycobacterium tuberculosis(M.TB). However, false-negative and false-positive results caused by base mismatches could affect the detection accuracy. Inspired by the unique property of CRISPR/Cas9, we proposed a new MSPQC M.TBsensor based on the CRISPR/Cas9 system, which can distinguish single-base mismatches in 10 bases from the protospacer adjacent motif (PAM) region. In the proposed sensor, single-stranded DNA on Au interdigital electrodes was used as a capture probe for the target and an initiator for hybridization chain reaction (HCR). HCR was used to generate long double-stranded DNA (dsDNA), which could span the Au interdigital electrodes. CRISPR/Cas9 was used as recognition components to recognize capture/target dsDNA. When the target existed, the capture probe hybridized with the target to form dsDNA, which could be recognized and cut by CRISPR/Cas9. Thus, the DNA connection between electrodes was cut off and resulted in the MSPQC response. When no target existed, the capture probe remained single-stranded and could not be recognized and cut by CRISPR/Cas9. Therefore, DNA connection between electrodes was reserved. Moreover, silver staining technology was utilized to improve the sensitivity of detection. M.TBwas detected by the proposed sensor using specific sequence fragments of 16S rRNA of M.TBas the target. The detection time was down to 2.3 h. The limit of detection (LOD) was 30 CFU/mL.