Detection of Frog Virus 3 by Integrating RPA-CRISPR/Cas12a-SPM with Deep Learning.

ABSTRACT

A fast, easy-to-implement, highly sensitive, and point-of-care (POC) detection system for frog virus 3 (FV3) is proposed. Combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a, a limit of detection (LoD) of 100 aM (60.2 copies/µL) is achieved by optimizing RPA primers and CRISPR RNAs (crRNAs). For POC detection, smartphone microscopy is implemented, and an LoD of 10 aM is achieved in 40 min. The proposed system detects four positive animal-derived samples with a quantitation cycle (Cq) value of quantitative PCR (qPCR) in the range of 13 to 32. In addition, deep learning models are deployed for binary classification (positive or negative samples) and multiclass classification (different concentrations of FV3 and negative samples), achieving 100 and 98.75% accuracy, respectively. Without temperature regulation and expensive equipment, the proposed RPA-CRISPR/Cas12a combined with smartphone readouts and artificial-intelligence-assisted classification showcases the great potential for FV3 detection, specifically POC detection of DNA virus.