CRISPR/Cas12b 辅助环介导等温扩增在一锅法中实现了对慢性 HBV DNA 的简便、快速和灵敏的定量。

CRISPR/Cas12b assisted loop-mediated isothermal amplification for easy, rapid and sensitive quantification of chronic HBV DNA in one-pot.

机构信息

The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, 350025, PR China; Mengchao Med-X Center, Fuzhou University, Fuzhou, 350116, PR China; College of Chemical Engineering, Fuzhou University, Fuzhou, 350116, PR China.

School of Basic Medical Sciences, Fujian Medical University, Fuzhou, 350122, PR China.

出版信息

Anal Chim Acta. 2024 Jun 29;1310:342702. doi: 10.1016/j.aca.2024.342702. Epub 2024 May 7.

DOI:10.1016/j.aca.2024.342702

PMID:38811141

Abstract

BACKGROUND

Currently, millions of people suffer from undiagnosed chronic hepatitis B (CHB) infection each year, which leads to high mortality rates attributed to cirrhosis and hepatocellular carcinoma. Previously reported assays, such as PCR-based assays, have limitations in terms of convenient for CHB screening in high-burden regions and resource-limited settings. Recently, diagnosis based on CRISPR/Cas, which has been considered as a potential method of point-of-care test (POCT) in resource-limited settings, offers a significant advantage in terms of high sensitivity and specificity. Therefore, there is an urgent need for the hepatitis B virus (HBV) detection utilizing CRISPR/Cas system.

RESULTS

We have proposed a one-pot of one-step method for CRISPR/Cas12b assisted loop-mediated isothermal amplification (LAMP) to facilitate the quick, sensitive, and precise quantification of HBV DNA. This method is designed for point-of-care testing following genomic extraction or sample heat treatment. We have optimized several critical factors, such as the reaction buffer, AapCas12b-gRNA concentration, reporter and its concentration, reaction temperature, and chemical additives, to significantly enhance the performance of the one-pot assay for HBV. Importantly, it exhibited no cross-reactivity between HBV and blood-borne pathogens. Moreover, the assay is capable of quantifying HBV DNA within 1 h with a limit of detection (LOD) of 25 copies per milliliter. Additionally, when tested on 236 clinical samples, the assay demonstrated a sensitivity of 99.00 % (198/200) and a specificity of 100.00 % (36/36) at the 99 % confidence level compared to real-time quantitative PCR.

SIGNIFICANCE

The utilization of convenient and reliable point-of-care diagnostic methods is crucial for reducing the burden of CHB globally. The assay we developed was helpful to improve the ability of HBV diagnosis for practical clinical translation, especially in high-burden regions and resource-limited settings. It has great advantages for rapid screening of CHB as well as evaluation of therapeutic efficacy as a companion diagnostic method.

摘要

背景

目前，每年都有数百万人患有未确诊的慢性乙型肝炎（CHB）感染，这导致肝硬化和肝细胞癌的死亡率很高。以前报道的检测方法，如基于 PCR 的检测方法，在高负担地区和资源有限的环境中进行 CHB 筛查方面存在局限性。最近，基于 CRISPR/Cas 的诊断方法被认为是资源有限环境中即时护理测试（POCT）的一种潜在方法，具有高灵敏度和特异性的显著优势。因此，迫切需要利用 CRISPR/Cas 系统检测乙型肝炎病毒（HBV）。

结果

我们提出了一种一步法的方法，用于 CRISPR/Cas12b 辅助环介导等温扩增（LAMP），以促进 HBV DNA 的快速、敏感和精确定量。该方法设计用于基因组提取或样品热处理后的即时护理测试。我们优化了几个关键因素，如反应缓冲液、AapCas12b-gRNA 浓度、报告分子及其浓度、反应温度和化学添加剂，显著提高了该方法检测 HBV 的性能。重要的是，该方法与血液传播病原体之间没有交叉反应。此外，该检测方法可以在 1 小时内定量检测 HBV DNA，检测限（LOD）为 25 拷贝/毫升。此外，当在 236 个临床样本上进行测试时，与实时定量 PCR 相比，该检测方法在 99%置信水平下的灵敏度为 99.00%（198/200），特异性为 100.00%（36/36）。