Zhou Xuan, Tang Honghua, Luo Gemiao, Zou Lintao, Liu Hangxi, Wen Piaoting, Yang Ruifu, Deng Zhongliang
Department of Public Health Laboratory Sciences, College of Public Health, Hengyang Medical School, University of South China, Hengyang, Hunan, China.
The First Affiliated Hospital of University of South China, Hengyang Medical School, University of South China, Hengyang, Hunan, China.
Anal Methods. 2025 Feb 13;17(7):1503-1510. doi: 10.1039/d4ay02079k.
Hepatitis B virus (HBV) is a major pathogen posing significant challenges to global public health, making early diagnosis critical for preventing severe liver diseases. We previously developed a fluorescent biosensor named PAM-dependent dsDNA Target-activated Cas12f1 Trans Reporter (PDTCTR). However, its reliance on specialized fluorescence equipment and lack of visual readout limited its application in resource-limited settings. To address these limitations, we developed a lateral flow assay (LFA) that integrates enzymatic recombinase amplification (ERA) with the Cas12f1_ge4.1 system. This approach enables the specific amplification of the HBV target gene through ERA and leverages the precise cleavage activity of Cas12f1_ge4.1 for enhanced signal amplification. The entire detection process is completed within 50 minutes, with results readily interpretable through visual inspection. The assay achieves a minimum detection limit of 100 copies per μL and demonstrates high specificity, showing no cross-reactivity with related viruses. In a validation study involving 71 clinical samples, the system achieved a sensitivity of 94.23%, specificity of 100%, and a kappa value of 0.90 compared to quantitative PCR (qPCR), indicating high reliability. This method thus shows promise as an effective tool for early HBV diagnosis, particularly suited for rapid, on-site detection in resource-constrained environments, and holds broad potential for diverse applications.
乙型肝炎病毒（HBV）是对全球公共卫生构成重大挑战的主要病原体，因此早期诊断对于预防严重肝脏疾病至关重要。我们之前开发了一种名为PAM依赖性双链DNA靶标激活的Cas12f1反式报告基因（PDTCTR）的荧光生物传感器。然而，其对专用荧光设备的依赖以及缺乏视觉读数限制了其在资源有限环境中的应用。为了解决这些限制，我们开发了一种将酶促重组酶扩增（ERA）与Cas12f1_ge4.1系统整合的侧向流动分析（LFA）。这种方法能够通过ERA对HBV靶基因进行特异性扩增，并利用Cas12f1_ge4.1的精确切割活性来增强信号放大。整个检测过程在50分钟内完成，结果通过目视检查即可轻松解读。该分析方法的最低检测限为每微升100拷贝，并具有高特异性，与相关病毒无交叉反应。在一项涉及71份临床样本的验证研究中，与定量PCR（qPCR）相比，该系统的灵敏度为94.23%，特异性为100%，kappa值为0.90，表明具有高可靠性。因此，该方法有望成为早期HBV诊断的有效工具，特别适用于资源有限环境中的快速现场检测，并具有广泛的应用潜力。
