Dual Detection of Hepatitis B and C Viruses Using CRISPR-Cas Systems and Lateral Flow Assay.

IF 1.3 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Journal of Nucleic Acids Pub Date : 2024-10-05 eCollection Date: 2024-01-01 DOI:10.1155/2024/8819834
Syeda Najidah Shahni, Sarah Albogami, Iqbal Azmi, Bijay Pattnaik, Rituparna Chaudhuri, Kapil Dev, Jawed Iqbal, Amit Sharma, Tanveer Ahmad
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Abstract

The development of sensitive and specific diagnostic tools for hepatitis B virus (HBV) and hepatitis C virus (HCV) remains crucial for effective disease management and control. In this study, we utilized CRISPR-Cas12 and CRISPR-Cas13 systems for the detection of HBV (DNA virus) and HCV (RNA virus), respectively. We designed and tested multiple guide RNAs (gRNAs) targeting both viruses, confirming successful cleavage of target sequences through gel electrophoresis and a fluorescent reporter assay. Using optimized gRNAs, we developed a lateral flow assay (LFA) for sensitive detection of HBV and HCV, demonstrating a concentration-dependent signal increase. Importantly, no cross-reactivity was observed with other viral targets. To further enhance sensitivity, we employed a dual-enzyme approach, combining Cas12 and Cas13 in a single reaction, which significantly improved detection limits for both viruses. Finally, we developed a dual antigen detection LFA strip capable of simultaneously detecting both HBV and HCV in a single sample. This approach holds promise for point-of-care (POC) diagnostics where the specific viral infection is unknown. This study addresses the current limitations in CRISPR-Cas based diagnostics, namely, the need for ultrasensitive detection methods and the ability to detect multiple antigens using a single test strip. Our findings demonstrate the feasibility of using CRISPR-Cas systems for highly sensitive and specific detection of HBV and HCV, paving the way for potential POC application.

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利用 CRISPR-Cas 系统和侧流检测法双重检测乙型肝炎和丙型肝炎病毒。
开发针对乙型肝炎病毒(HBV)和丙型肝炎病毒(HCV)的敏感而特异的诊断工具对于有效的疾病管理和控制仍然至关重要。在本研究中,我们利用 CRISPR-Cas12 和 CRISPR-Cas13 系统分别检测了 HBV(DNA 病毒)和 HCV(RNA 病毒)。我们设计并测试了多种针对这两种病毒的引导 RNA(gRNA),通过凝胶电泳和荧光报告实验证实了目标序列的成功裂解。利用优化的 gRNA,我们开发了一种横向流动检测法(LFA),用于灵敏检测 HBV 和 HCV,结果表明信号增加与浓度有关。重要的是,没有观察到与其他病毒靶点的交叉反应。为了进一步提高灵敏度,我们采用了一种双酶方法,将 Cas12 和 Cas13 结合在一个反应中,从而大大提高了对这两种病毒的检测限。最后,我们开发了一种双抗原检测 LFA 试剂条,能够在单一样本中同时检测 HBV 和 HCV。这种方法有望用于特异性病毒感染未知的床旁诊断(POC)。本研究解决了目前基于 CRISPR-Cas 的诊断方法存在的局限性,即需要超灵敏的检测方法以及使用单个试纸检测多种抗原的能力。我们的研究结果证明了使用 CRISPR-Cas 系统对 HBV 和 HCV 进行高灵敏度和特异性检测的可行性,为潜在的 POC 应用铺平了道路。
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来源期刊
Journal of Nucleic Acids
Journal of Nucleic Acids BIOCHEMISTRY & MOLECULAR BIOLOGY-
CiteScore
3.10
自引率
21.70%
发文量
5
审稿时长
12 weeks
期刊最新文献
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