Center of Precision Medicine and Healthcare, Tsinghua-Berkeley Shenzhen Institute; Institute of Biopharmaceutics and Health Engineering, Tsinghua Shenzhen International Graduate School.
Institute of Biopharmaceutics and Health Engineering, Tsinghua Shenzhen International Graduate School.
J Vis Exp. 2024 May 10(207). doi: 10.3791/64833.
We report a fast, easy-to-implement, highly sensitive, sequence-specific, and point-of-care (POC) DNA virus detection system, which combines recombinase polymerase amplification (RPA) and CRISPR/Cas12a system for trace detection of DNA viruses. Target DNA is amplified and recognized by RPA and CRISPR/Cas12a separately, which triggers the collateral cleavage activity of Cas12a that cleaves a fluorophore-quencher labeled DNA reporter and generalizes fluorescence. For POC detection, portable smartphone microscopy is built to take fluorescent images. Besides, deep learning models for binary classification of positive or negative samples, achieving high accuracy, are deployed within the system. Frog virus 3 (FV3, genera Ranavirus, family Iridoviridae) was tested as an example for this DNA virus POC detection system, and the limits of detection (LoD) can achieve 10 aM within 40 min. Without skilled operators and bulky instruments, the portable and miniature RPA-CRISPR/Cas12a-SPM with artificial intelligence (AI) assisted classification shows great potential for POC DNA virus detection and can help prevent the spread of such viruses.
我们报告了一种快速、易于实施、高度敏感、序列特异性和即时（POC）的 DNA 病毒检测系统，该系统结合了重组酶聚合酶扩增（RPA）和 CRISPR/Cas12a 系统，用于痕量 DNA 病毒的检测。靶 DNA 分别通过 RPA 和 CRISPR/Cas12a 进行扩增和识别，这触发了 Cas12a 的旁切活性，该活性切割荧光团猝灭剂标记的 DNA 报告分子并产生荧光。对于 POC 检测，构建了便携式智能手机显微镜来拍摄荧光图像。此外，还在系统内部署了用于阳性或阴性样本的二进制分类的深度学习模型，实现了高精度。以蛙病毒 3（FV3，虹彩病毒目，虹彩病毒科）为例，对该 DNA 病毒 POC 检测系统进行了测试，其检测限（LoD）在 40 分钟内可达到 10 aM。该便携式和微型的 RPA-CRISPR/Cas12a-SPM 具有人工智能（AI）辅助分类功能，无需熟练操作人员和庞大的仪器，具有用于 POC DNA 病毒检测的巨大潜力，并有助于防止此类病毒的传播。
