<br>CRISPR/Cas12b 辅助环介导等温扩增，一锅法轻松、快速、灵敏地定量慢性 HBV DNA,Analytica Chimica Acta

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CRISPR/Cas12b 辅助环介导等温扩增，一锅法轻松、快速、灵敏地定量慢性 HBV DNA
Analytica Chimica Acta ( IF 5.7 ) Pub Date : 2024-05-07 , DOI: 10.1016/j.aca.2024.342702
Haipo Xu ₁ , Gengping Lin ₂ , Ronghua Chen ₃ , Zhixiong Cai ₃ , Yupeng Sun ₃ , Xiaolong Zhang ₃ , Bixing Zhao ₃ , Yongyi Zeng ₃ , Jingfeng Liu ₄ , Xiaolong Liu ₅

Affiliation

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.
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.
Mengchao Med-X Center, Fuzhou University, Fuzhou, 350116, PR China; College of Chemical Engineering, Fuzhou University, Fuzhou, 350116, PR China; Department of Radiation Oncology, Fujian Medical University Cancer Hospital, Fuzhou, 350014, PR China.
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; School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, 710049, PR China.

目前，每年有数百万人患有未确诊的慢性乙型肝炎（CHB）感染，导致肝硬化和肝细胞癌的高死亡率。先前报道的检测方法，例如基于 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。近年来，基于CRISPR/Cas的诊断被认为是在资源有限的情况下进行即时检测（POCT）的潜在方法，在高灵敏度和特异性方面具有显着优势。因此，迫切需要利用CRISPR/Cas系统进行乙型肝炎病毒（HBV）检测。我们提出了一种 CRISPR/Cas12b 辅助环介导等温扩增 (LAMP) 的一锅一步法，以促进 HBV DNA 的快速、灵敏和精确定量。该方法专为基因组提取或样品热处理后的现场检测而设计。我们优化了反应缓冲液、AapCas12b-gRNA浓度、报告基因及其浓度、反应温度和化学添加剂等几个关键因素，以显着提高乙型肝炎病毒一锅法检测的性能。重要的是，它没有表现出乙型肝炎病毒和血源性病原体之间的交叉反应。此外，该检测能够在 1 小时内定量 HBV DNA，检测限 (LOD) 为每毫升 25 个拷贝。此外，在对 236 个临床样本进行测试时，与实时定量 PCR 相比，该检测方法的灵敏度为 99.00% (198/200)，特异性为 100.00% (36/36)，置信水平为 99%。使用方便可靠的即时诊断方法对于减轻全球慢性乙型肝炎的负担至关重要。我们开发的检测方法有助于提高乙型肝炎诊断的实际临床转化能力，特别是在高负担地区和资源有限的地区。作为伴随诊断方法，它对于慢性乙型肝炎的快速筛查以及疗效评价具有巨大的优势。

"点击查看英文标题和摘要"

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

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. 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. 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.

更新日期：2024-05-07

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