Gu Xijuan, Pan Anli, Wu Lingwei, Zhang Jing, Xu Zixun, Wen Tao, Wang Miaomiao, Shi Xiuying, Wu Li, Qin Yuling
Nantong Key Laboratory of Public Health and Medical Analysis, School of Public Health, Nantong University Nantong Jiangsu 226019 P. R. China
Xinglin College, Nantong University Qidong Jiangsu 226236 P. R. China.
Chem Sci. 2024 Oct 8;15(44):18411-8. doi: 10.1039/d4sc05226a.
Rapid and precise detection of respiratory pathogens is crucial for clinical diagnosis and treatment of respiratory infections. In this study, the multiplex and visual detection of respiratory pathogens is facilitated by specifically designed engineered CRISPR RNA (en-crRNA) to activate the -cleavage activity of Cas12a, along with a homemade portable device. The en-crRNA comprised an original crRNA and a DNA reporter molecule that is labelled with both a fluorophore and a quencher. Moreover, the DNA is partially complementary to the variable region of the original crRNA. The proof of concept was demonstrated by simultaneously identifying distinct respiratory pathogens with a detection limit of 10 copies per μL. The visual discrimination was subsequently achieved using a homemade portable device that was seamlessly integrated with a smartphone. The specificity of the strategy was validated by comparing with qPCR assays for clinical sample detection, demonstrating exceptional accuracy with areas under the ROC curves of 0.98 for all targets. The research provides a promising avenue for the development of rapid, specific, and on-site detection techniques aimed at multiplex identification of respiratory pathogens.
快速准确地检测呼吸道病原体对于呼吸道感染的临床诊断和治疗至关重要。在本研究中，通过专门设计的工程化CRISPR RNA（en-crRNA）激活Cas12a的切割活性，并结合自制便携式设备，实现了呼吸道病原体的多重可视化检测。en-crRNA由原始crRNA和一个同时标记有荧光团和淬灭剂的DNA报告分子组成。此外，该DNA与原始crRNA的可变区部分互补。通过同时鉴定不同的呼吸道病原体，检测限为每微升10个拷贝，证明了概念验证。随后使用与智能手机无缝集成的自制便携式设备实现了视觉辨别。通过与用于临床样本检测的qPCR分析进行比较，验证了该策略的特异性，所有目标的ROC曲线下面积为0.98，证明了其卓越的准确性。该研究为开发旨在多重鉴定呼吸道病原体的快速、特异和现场检测技术提供了一条有前景的途径。
