Institute of Pediatrics, Shenzhen Children's Hospital, Clinical Medical College of Southern University of Science and Technology, Shenzhen, 518038, Guangdong, PR China; Institute of Biomedical and Health Engineering, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, Guangdong, PR China.
Interdisciplinary Center of High Magnetic Field Physics of Shenzhen University, College of Physics and Optoelectronic Engineering, Institute of Microscale Optoelectronics, Shenzhen University, Shenzhen, 518060, PR China; Shenzhen International Institute for Biomedical Research, 3/F, Building 1-B, Silver Star Hi-tech Industrial Park, Longhua District, Shenzhen, 518110, PR China.
Biosens Bioelectron. 2023 Oct 1;237:115523. doi: 10.1016/j.bios.2023.115523. Epub 2023 Jul 9.
Respiratory viruses have long been a major cause of a global pandemic, emphasizing the urgent need for high-sensitivity diagnostic tools. Typical PCR technology can only determine the type of virus in the sample, which is unable to detect different variants of the same virus without costly and time-consuming gene sequencing. Here, we introduce a simple, fully enclosed, and highly integrated microfluidic system based on CRISPR/Cas12a and isothermal amplification techniques (LOC-CRISPR) that can specifically identify multiple common respiratory viruses and their variants. The LOC-CRISPR chip integrates viral nucleic acid extraction, recombinant polymerase amplification, and CRISPR/Cas12a cleavage reaction-based detection, contamination-free detection. In addition, the LOC-CRISPR chip was designed for multiplexed detection (two-sample input and ten-result outputs), which can not only detect the presence of SARS-CoV-2, HN, HN, IVB and HRSV but also differentiate the BA.1, BA.2, and BA.5 variants of SARS-COV-2. For clinical validation, the LOC-CRISPR chip was used to analyze 50 nasopharyngeal swab samples (44 positive and 6 negative) and achieved excellent sensitivity (97.8%) and specificity (100%). This innovative LOC-CRISPR system has the ability to quickly, sensitively, and accurately detect multiple target nucleic acid sequences with single-base mutations, which will further improve the rapid identification and traceability of respiratory viruses infectious diseases.
呼吸道病毒长期以来一直是引发全球大流行的主要原因，这强调了对高灵敏度诊断工具的迫切需求。典型的 PCR 技术只能确定样本中的病毒类型，无法在不进行昂贵且耗时的基因测序的情况下检测到同一病毒的不同变体。在这里，我们介绍了一种简单、完全封闭且高度集成的微流控系统，该系统基于 CRISPR/Cas12a 和等温扩增技术（LOC-CRISPR），可特异性识别多种常见呼吸道病毒及其变体。LOC-CRISPR 芯片集成了病毒核酸提取、重组聚合酶扩增和基于 CRISPR/Cas12a 切割反应的检测，实现了无污染检测。此外，LOC-CRISPR 芯片设计用于多重检测（两个样本输入和十个结果输出），不仅可以检测 SARS-CoV-2、HN、IVB 和 HRSV 的存在，还可以区分 SARS-CoV-2 的 BA.1、BA.2 和 BA.5 变体。为了进行临床验证，我们使用 LOC-CRISPR 芯片分析了 50 份鼻咽拭子样本（44 份阳性和 6 份阴性），并取得了优异的灵敏度（97.8%）和特异性（100%）。这种创新的 LOC-CRISPR 系统能够快速、灵敏、准确地检测具有单碱基突变的多个目标核酸序列，这将进一步提高呼吸道病毒传染病的快速识别和溯源能力。
