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

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Development of an MSPQC Nucleic Acid Sensor Based on CRISPR/Cas9 for the Detection of Mycobacterium tuberculosis
Analytical Chemistry ( IF 6.7 ) Pub Date : 2022-08-05 , DOI: 10.1021/acs.analchem.2c02538
Ji Huang ₁ , Zi Liang ₁ , Yu Liu ₁ , Jiandang Zhou ₂ , Fengjiao He ₁

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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.TB 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. M.TB was detected by the proposed sensor using specific sequence fragments of 16S rRNA of M.TB as the target. The detection time was down to 2.3 h. The limit of detection (LOD) was 30 CFU/mL.

中文翻译：

基于 CRISPR/Cas9 的 MSPQC 核酸传感器开发用于结核分枝杆菌检测

核酸的准确、快速检测在结核分枝杆菌（M.TB）所致结核病的临床治疗中发挥着至关重要的作用。然而，由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 独特特性的启发，我们提出了一种新的 MSPQC M.TB基于 CRISPR/Cas9 系统的传感器，可以区分 10 个碱基中的单碱基错配与 protospacer 相邻基序 (PAM) 区域。在所提出的传感器中，Au 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) 的引发剂。HCR 用于生成长双链 DNA (dsDNA)，它可以跨越 Au 叉指电极。CRISPR/Cas9 被用作识别组件以识别捕获/目标 dsDNA。当靶标存在时，捕获探针与靶标杂交形成dsDNA，可以被CRISPR/Cas9识别和切割。因此，电极之间的 DNA 连接被切断并导致 MSPQC 响应。当目标不存在时，捕获探针仍然是单链的，不能被 CRISPR/Cas9 识别和切割。因此，保留了电极之间的 DNA 连接。此外，银染色技术被用来提高检测的灵敏度。所提出的传感器使用 M.TB 的 16S rRNA 的特定序列片段作为目标来检测M.TB。检测时间降至 2.3 h。检测限 (LOD) 为 30 CFU/mL。

更新日期：2022-08-05

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