DNA Virus Detection System Based on RPA-CRISPR/Cas12a-SPM and Deep Learning.

Journal: Journal of visualized experiments : JoVE
PMID: 38801262

Abstract

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.

Authors

  • Changyue Liu
    Center of Precision Medicine and Healthcare, Tsinghua-Berkeley Shenzhen Institute; Institute of Biopharmaceutics and Health Engineering, Tsinghua Shenzhen International Graduate School.
  • Zhengyang Lei
    Institute of Biopharmaceutical and Health Engineering, Shenzhen International Graduate School, Tsinghua University, Shenzhen, Guangdong 518055, China; Precision Medicine and Public Health, Tsinghua-Berkeley Shenzhen Institute, Tsinghua University, Shenzhen, Guangdong 518055, China.
  • Lijin Lian
    Institute of Biopharmaceutical and Health Engineering, Shenzhen International Graduate School, Tsinghua University, Shenzhen, Guangdong 518055, China; Precision Medicine and Public Health, Tsinghua-Berkeley Shenzhen Institute, Tsinghua University, Shenzhen, Guangdong 518055, China.
  • Likun Zhang
    Center of Precision Medicine and Healthcare, Tsinghua-Berkeley Shenzhen Institute; Institute of Biopharmaceutics and Health Engineering, Tsinghua Shenzhen International Graduate School.
  • Zhicheng Du
    Department of Medical Statistics and Epidemiology & Health Information Research Center & Guangdong Key Laboratory of Medicine, School of Public Health, Sun Yat-sen University, Guangzhou 510080, China.
  • Peiwu Qin
    Institute of Biopharmaceutical and Health Engineering, Shenzhen International Graduate School, Tsinghua University, Shenzhen, Guangdong 518055, China; Precision Medicine and Public Health, Tsinghua-Berkeley Shenzhen Institute, Tsinghua University, Shenzhen, Guangdong 518055, China. Electronic address: pwqin@sz.tsinghua.edu.cn.

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