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

Abstract

Accurate and rapid detection of nucleic acid plays a vital role in the clinical treatment of tuberculosis caused by <i>Mycobacterium tuberculosis</i> (<i>M.TB</i>). 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 <i>M.TB</i> sensor 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. <i>M.TB</i> was detected by the proposed sensor using specific sequence fragments of 16S rRNA of <i>M.TB</i> as the target. The detection time was down to 2.3 h. The limit of detection (LOD) was 30 CFU/mL.