Wu Junhua, Yan Qiyan, Qiu Haiyan, Gao E-Bin
Department of Pediatrics, The Affiliated Women and Children's Hospital of Ningbo University, Ningbo, China.
School of Life Sciences, Jiangsu University, Zhenjiang, China.
J Med Virol. 2025 Jun;97(6):e70426. doi: 10.1002/jmv.70426.
Human norovirus (NoV) is a primary cause of acute gastroenteritis in children, making accurate and rapid detection essential for effective disease prevention and control. In this study, we developed a sensitive and efficient platform for pathogen nucleic acid detection by integrating asymmetric nucleic acid sequence-based amplification (asymmetric NASBA), enzyme-DNA molecular complex, and the clustered regularly interspaced short palindromic repeats (CRISPR) system, namely an A-enDMC platform. The target recognition capability of the enzyme-DNA complex operates independently from the signal amplification function of the CRISPR system. By decoupling the CRISPR reaction from the dependence on specific target sequences, the platform's universality and modularity are enhanced. The assay is fast (< 1.5 h), highly sensitive (< 5 copies/µL), and demonstrates no cross-reactivity with other common viruses. Compared to the widely used RT-qPCR method, the platform demonstrates high consistency in detection results, with the detection coincidence rate of 96.77% and a kappa value of 0.87. This platform provides a versatile technological tool for highly sensitive and specific RNA detection, demonstrating its extensive potential in real sample analysis.
人诺如病毒（NoV）是儿童急性胃肠炎的主要病因，因此准确快速的检测对于有效的疾病预防和控制至关重要。在本研究中，我们通过整合基于不对称核酸序列的扩增（asymmetric NASBA）、酶-DNA分子复合物和规律成簇间隔短回文重复序列（CRISPR）系统，开发了一种灵敏高效的病原体核酸检测平台，即A-enDMC平台。酶-DNA复合物的靶标识别能力独立于CRISPR系统的信号放大功能。通过将CRISPR反应与对特定靶标序列的依赖性解耦，增强了平台的通用性和模块化。该检测方法快速（<1.5小时）、高度灵敏（<5拷贝/微升），且与其他常见病毒无交叉反应。与广泛使用的RT-qPCR方法相比，该平台检测结果具有高度一致性，检测符合率为96.77%，kappa值为0.87。该平台为高灵敏和特异的RNA检测提供了一种通用的技术工具，在实际样品分析中显示出广泛的潜力。
