School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, China.
State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China.
Front Cell Infect Microbiol. 2022 Mar 2;12:835213. doi: 10.3389/fcimb.2022.835213. eCollection 2022.
has gradually become a common pathogen in clinical microbial infections. Identification of at the species level is essential to assess the susceptibility and pathogenicity of antimicrobials. However, there is no suitable method for rapid and accurate laboratory detection of . In this study, we combined PCR amplification with the CRISPR-Cas12a system to establish a novel detection platform, named CRISPR-PCR, and applied it to the detection of in clinical samples. The Cas12a protein exhibited collateral cleavage activity following CRISPR RNA binding to specific targets, then indiscriminately cleaved nearby single-stranded DNA, and this was evaluated for diagnostic nucleic acid detection by measuring the fluorescence signal using a fluorescence reader. The assay takes only 2 h, including DNA extraction for 20 min, nucleic acid pre-amplification for 70 min, and fluorescence detection for 20 min. The limit of detection for was 10 ng and the specificity was 100%. Thus, the CRISPR-PCR assay is a rapid and specific method for detecting , and the CRISPR-PCR fluorescence detection platform has great potential for detection of other pathogens.
已逐渐成为临床微生物感染的常见病原体。在评估抗菌药物的敏感性和致病性方面，对 的种属水平进行鉴定至关重要。然而，目前尚无合适的方法可用于 的快速、准确的实验室检测。在本研究中，我们将 PCR 扩增与 CRISPR-Cas12a 系统相结合，建立了一种新型的检测平台，命名为 CRISPR-PCR，并将其应用于临床样本中 的检测。Cas12a 蛋白在与特定靶标结合的 CRISPR RNA 后表现出旁切活性，然后随机切割附近的单链 DNA，通过使用荧光读取器测量荧光信号来评估诊断核酸检测。该测定仅需 2 小时，包括 20 分钟的 DNA 提取、70 分钟的核酸预扩增和 20 分钟的荧光检测。 的检测限为 10ng，特异性为 100%。因此，CRISPR-PCR 检测法是一种快速、特异的检测 方法，CRISPR-PCR 荧光检测平台在检测其他病原体方面具有很大的潜力。
Zotero 插件
