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

Summary

We present a protocol that combines recombinase polymerase amplification with a CRISPR/Cas12a system for trace detection of DNA viruses and builds portable smartphone microscopy with an artificial intelligence-assisted classification for point-of-care DNA virus detection.

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.

Introduction

In recent years, epidemics of infectious diseases caused by different viruses have occurred frequently, including the Ebola virus disease (EVD) epidemic in 2014¹ and 2018², the Middle East Respiratory Syndrome (MERS) in 2015³, the Zika virus disease epidemic in 2015⁴, the Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)⁵ and the continuing Monkeypox caused by Monkeypox virus (MKPV) in 2022⁶. These sudden outbreaks of epidemic infectious diseases cause a large number of ....

Protocol

1. Processing of samples

1. Take Frog Virus 3 (FV3, genera Ranavirus, family Iridoviridae), a double-stranded DNA virus. Select the major capsid (mcp) gene as the target for the detection of FV3 since it is highly conserved and usually regarded as the target for ranavirus detection. The target sequence selected is shown in Table 1.
   NOTE: Frog Virus 3 is taken as an example in this protocol.
2. For target DNA fragments preparation, use DNA fragments of the mcp gene from FV3 and Infectious Spleen and Kidney Necrosis Virus (ISKNV, another Ranavirus).
   NOTE: In this study, t....

Results

This method focuses on a fast, easy-to-implement, highly sensitive, and point-of-care (POC) detection system for DNA viruses. Primer pairs design for the RPA reaction and crRNA design for CRISPR/Cas12a reaction are two of the essential parts since they will affect the efficiency of the RPA-CRISPR/Cas12a reaction and influence the subsequent detection and classification.

In this method, FV3 is regarded as an example of DNA virus detection. Some RPA primer pairs for FV3 are.......

Discussion

In this method, we develop a fast, easy-to-implement, highly sensitive, sequence-specific, and POC DNA virus detection system with AI assistance. After obtaining samples, RPA is applied to amplify the target sequence, and then CRISPR/Cas12a can recognize the target DNA and release fluorescence, which enlarges the detection signal. Portable smartphone microscopy is built to take fluorescence images, and deep learning models with transfer learning are used for binary classification of the positive and negative samples' ima.......

Materials

Name	Company	Catalog Number	Comments
20x amplification	OLYMPUS	OPLN20X
532 nm green laser	Thorlabs	PL201	with 0.9 mW output power
535 nm cutoff wavelength	chrome	AT535
6x DNA loading buffer	Thermo scientific	R0611
96-well black microplate	Corning Incorporated	3603	Black with flat clear bottom
Aspherical lens	Lubang	N/A
Bandpass filter	SEMROCK	FF01-542/27-25
Bsu DNA Polymerase	ATG Biotechnology	M103	Large Fragment
crRNA	Sangon Biotech	N/A
DNA fragments	Sangon Biotech	N/A
Dichroic holders	Ruicage	N/A
Dichroic mirror	SEMROCK	FF555-Di03-25x36	with a cutoff wavelength of 535 nm
E.Z.N.A Gel Extraction Kit	Omega Biotek	D2500-02
EnGen Lba Cas12a (Cpf1)	New England Biolabs (Beijing) LTD	M0653T
Filter holders	Ruicage	N/A
Fluorophore-ssDNA-Quencher reporter probes	Sangon Biotech	N/A	TAMRA (carboxy tetramethylrhodamine) as the fluorophore at the 5 ends; BHQ2 (Black Hole Quencher-2) as the quencher at the 3 ends
GP32	ATG Biotechnology	M104
ImageJ	Open-source	Version 1.53t 24	Downloaded from https://imagej.nih.gov/ij/
Microplate reader	SPARK, TECAN	N/A
Multi-Block thermal Cycler PCR instrument	LongGene	N/A
NanoDrop 2000/2000c Spectrophotometers	Thermo Scientific	ND-2000
NEBuffer r2.1	New England Biolabs (Beijing) LTD	B6002S	10x CRISPR/Cas12a Reaction buffer
Oxygen plasma treatment	Electro-Technic  Products	N/A
Pathogen Inactivate, Nucleic acid extraction-free, Direct-to-PCR Buffer with Proteinase K (PINDBK)	Ebio	PINDBK -25mL
PCR primer pairs	Sangon Biotech	N/A
PDMS	Dow Corning	Sylgard 184
RPA primer pairs	Sangon Biotech	N/A
Smartphone	Huawei	Mate10
Translation stages	Ruicage	N/A
Transmitted neutral density filters	Thorlabs	ND40A
Triplet achromatic lenses	Thorlabs	TRH127-020-A
UvsX	ATG Biotechnology	M105
UvsY	ATG Biotechnology	M106