Recombinase Polymerase Amplification and Target-Triggered CRISPR/Cas12a Assay for Sensitive and Selective Hepatitis B Virus DNA Analysis Based on Lanthanide Tagging and Inductively Coupled Plasma Mass Spectrometric Detection.

IF 6.7 1区化学 Q1 CHEMISTRY, ANALYTICAL Analytical Chemistry Pub Date : 2024-09-17 Epub Date: 2024-09-06 DOI:10.1021/acs.analchem.4c03715

Chenxi Zhao, Lijie Du, Jing Hu, Xiandeng Hou

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Abstract

Herein, we report a target-triggered CRISPR/Cas12a assay by coupling lanthanide tagging and inductively coupled plasma mass spectrometry (ICP-MS) for highly sensitive elemental detection. Hepatitis B virus (HBV) DNA was chosen as a model analyte, and recombinase polymerase amplification (RPA) was used for target amplification. The double-stranded RPA amplicons containing a 5' TTTG PAM sequence can be recognized by Cas12a through a specific CRISPR RNA, activating the trans-cleavage activity of CRISPR/Cas12a and nonspecific cleavage of terbium (Tb)-ssDNA modified on magnetic beads (MBs). Following magnetic separation and acid digestion, the released Tb³⁺ ions were quantitated by ICP-MS and correlated to the concentration of HBV DNA. Taking advantage of the accelerated cleavage of Tb-ssDNA attached to the MB particles, RPA for target amplification, and ICP-MS for highly selective signal readout, this method permits the detection of 1 copy/μL of HBV DNA in serum with high specificity and holds great promise in the early diagnosis of viral infections or tumor development.

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基于镧系元素标记和电感耦合等离子体质谱检测的重组酶聚合酶扩增和靶标触发 CRISPR/Cas12a 分析法，用于灵敏、选择性地分析乙型肝炎病毒 DNA。

在此，我们报告了一种靶标触发的 CRISPR/Cas12a 检测方法，该方法将镧系元素标记与电感耦合等离子体质谱（ICP-MS）联用，实现了高灵敏度的元素检测。该方法以乙型肝炎病毒（HBV）DNA 为模型分析物，采用重组酶聚合酶扩增（RPA）技术进行目标扩增。含有 5' TTTG PAM 序列的双链 RPA 扩增子可通过特异性 CRISPR RNA 被 Cas12a 识别，激活 CRISPR/Cas12a 的反式裂解活性，并非特异地裂解修饰在磁珠（MB）上的铽（Tb）-ssDNA。磁分离和酸消化后，释放的 Tb3+ 离子通过 ICP-MS 进行定量，并与 HBV DNA 的浓度相关联。利用磁珠颗粒上附着的 Tb-ssDNA 的加速裂解、RPA 的目标扩增和 ICP-MS 的高选择性信号读出，该方法可以高特异性地检测血清中 1 拷贝/μL 的 HBV DNA，在病毒感染或肿瘤发展的早期诊断中大有可为。

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来源期刊

Analytical Chemistry 化学-分析化学

CiteScore

12.10

自引率

12.20%

发文量

1949

审稿时长

1.4 months

期刊介绍： Analytical Chemistry, a peer-reviewed research journal, focuses on disseminating new and original knowledge across all branches of analytical chemistry. Fundamental articles may explore general principles of chemical measurement science and need not directly address existing or potential analytical methodology. They can be entirely theoretical or report experimental results. Contributions may cover various phases of analytical operations, including sampling, bioanalysis, electrochemistry, mass spectrometry, microscale and nanoscale systems, environmental analysis, separations, spectroscopy, chemical reactions and selectivity, instrumentation, imaging, surface analysis, and data processing. Papers discussing known analytical methods should present a significant, original application of the method, a notable improvement, or results on an important analyte.