State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China.
Department of Clinical Laboratory, The Third Xiangya Hospital, Xiangya Medical College of Central South University, Changsha 410208, P. R. China.
Anal Chem. 2022 Aug 16;94(32):11409-11415. doi: 10.1021/acs.analchem.2c02538. Epub 2022 Aug 5.
Accurate and rapid detection of nucleic acid plays a vital role in the clinical treatment of tuberculosis caused by (). 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 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. was detected by the proposed sensor using specific sequence fragments of 16S rRNA of as the target. The detection time was down to 2.3 h. The limit of detection (LOD) was 30 CFU/mL.
准确、快速地检测核酸在结核病的临床治疗中起着至关重要的作用。然而，碱基错配引起的假阴性和假阳性结果可能会影响检测的准确性。受 CRISPR/Cas9 独特性质的启发，我们提出了一种基于 CRISPR/Cas9 系统的新型 MSPQC 传感器，它可以区分 PAM 区 10 个碱基内的单碱基错配。在提出的传感器中，金叉指电极上的单链 DNA 被用作目标的捕获探针和杂交链式反应（HCR）的起始物。HCR 用于生成可以跨越金叉指电极的长双链 DNA（dsDNA）。CRISPR/Cas9 被用作识别组件来识别捕获/靶标 dsDNA。当存在靶标时，捕获探针与靶标杂交形成 dsDNA，可被 CRISPR/Cas9 识别和切割。因此，电极之间的 DNA 连接被切断，产生 MSPQC 响应。当不存在靶标时，捕获探针保持单链状态，不能被 CRISPR/Cas9 识别和切割。因此，电极之间的 DNA 连接得以保留。此外，还利用银染技术提高了检测的灵敏度。使用 作为目标的 16S rRNA 的特定序列片段来检测 。检测时间缩短至 2.3 小时。检测限（LOD）为 30 CFU/mL。
