Rapid detection of African swine fever virus using Cas12a-based portable paper diagnostics

African swine fever (ASF) is a rapidly spreading, highly lethal disease of swine with major economic impact. With no effective vaccines or therapies available, control relies on rapid detection and culling to prevent spread. Existing OIE-recommended diagnostics (virus isolation, FAT, conventional/qPCR) are sensitive but require laboratory infrastructure, trained personnel, and instruments, making them less suitable for field deployment. The study aims to develop a rapid, specific, sensitive, and equipment-free assay for ASFV detection that is practical for on-site use by integrating CRISPR-Cas12a detection with a portable lateral-flow readout.

Traditional ASFV diagnostics include virus isolation (gold standard but slow and complex), antigen detection (FAT; scalable but insufficiently sensitive early in infection), and PCR/qPCR (fast and sensitive but instrument-dependent). Isothermal amplification methods enable constant-temperature DNA amplification but can suffer from nonspecific amplification and false positives. CRISPR-based diagnostics leverage cis-cleavage target recognition and collateral trans-cleavage of nucleic acids by enzymes such as Cas13a (RNA-guided, requires in vitro transcription and RNA reporters) and Cas12a (DNA-guided, cleaves ssDNA reporters). Prior Cas13a platforms have achieved attomolar sensitivity and can be coupled to lateral flow but are time-consuming and costlier due to RNA reagents. Cas12a-based assays are simpler and use stable, low-cost DNA reporters, but fluorescence readouts typically require instruments or blue-light visualization that are not field-friendly. This work builds on Cas12a’s collateral activity and integrates it with isothermal amplification (RPA) and gold nanoparticle-based lateral flow to create a portable, instrument-free ASFV detection platform.

- Target selection and crRNA design: Designed six crRNAs targeting highly conserved regions of the essential ASFV genes DNA polymerase (DNA Pol) and polyprotein pp220. Verified specific cis-cleavage of target dsDNA by Cas12a-crRNA complexes using in vitro cleavage assays. - Fluorescence reporter assay: Used an ssDNA FAM-quencher reporter (FAM-TTATT-Quencher). Upon Cas12a activation by target dsDNA, collateral cleavage of the reporter releases fluorescence. Screened crRNAs and identified DNA Pol crRNA1 and pp220 crRNA5 as top performers based on fluorescence intensity and kinetics. - Specificity and mismatch tolerance: Introduced systematic 2-nt and 1-nt mutations across PAM and PAM-proximal/distal target regions for crRNA1 and crRNA5. Assessed fluorescence to determine tolerance. Also tested 1-nt insertions/deletions. Evaluated cross-reactivity against genomes of common swine pathogens (PRV, PCV, Streptococcus suis, Mycoplasma hyorhinis, Haemophilus parasuis) and performed BLAST analyses to assess sequence similarity with human/suspected contaminant viruses. - Veterinary samples, fluorescence readout: Extracted DNA from blood and anal swabs of ASFV-infected and uninfected pigs. Enriched crRNA target regions by PCR, then incubated amplicons with Cas12a/crRNA and ssDNA-FQ reporter. Compared results with qPCR determinations. - Instrument-free platform (Cas-gold): Replaced PCR with recombinase polymerase amplification (RPA) for isothermal amplification and replaced fluorescence readout with a lateral flow strip engineered in-house. Constructed strips with gold nanoparticles conjugated to anti-FITC antibody on the conjugate pad; streptavidin (control line) and IgG (test line) immobilized on NC membrane. Used a FAM-Biotin ssDNA reporter that binds Au–anti-FITC conjugates. Intact reporter complexes are captured at the streptavidin control line; Cas12a-mediated cleavage releases biotin, allowing complexes to pass control and bind at the IgG test line. - Optimization of lateral flow: Tuned reporter concentration to eliminate false-positive test bands in blanks (settled on 1 μM FAM-Biotin reporter). Increased LbCas12a/crRNA concentration to sharpen positive test bands. Optimized reaction time (1–2 min sufficient) and temperature (room temperature superior to 4 °C or 37 °C) for lateral-flow development. - Analytical sensitivity: Tested serial dilutions of ASFV genome (1–10^5 copies) using RPA + Cas12a + lateral flow. Also assessed use of dual crRNAs (crRNA1 + crRNA5) for potential sensitivity gains. - Clinical/field evaluation: Tested 30 veterinary samples (blood, oral, anal swabs) with Cas-gold and compared against qPCR results and quantified viral loads for select samples.

- Lead crRNAs: DNA Pol crRNA1 and pp220 crRNA5 exhibited the strongest Cas12a activation in fluorescence assays, outperforming other designs. - High specificity and low mismatch tolerance: Mutations in PAM or PAM-proximal regions abolished or markedly reduced signal; only PAM-distal mutations (positions ~17–20) retained substantial activity. Some single-nucleotide mutations at select distal positions produced above-background signal, indicating partial tolerance. 1-nt insertions/deletions yielded little signal. - No cross-reactivity: Strong signal obtained only with ASFV DNA; PRV, PCV, SS, MH, and HPS produced few/no signals in both fluorescence and lateral flow. BLAST showed <60% similarity to non-ASFV viruses for guide regions. - Veterinary samples detected: Fluorescence assay correctly identified qPCR-positive blood (samples 1–5) and anal swab (sample 1) and showed no signal for qPCR-negative samples (blood 6–8; swabs 2–3), indicating high accuracy. - Portable Cas-gold assay performance: Lateral flow optimization eliminated false positives in blanks at 1 μM reporter and enhanced test band clarity with increased LbCas12a/crRNA. A clear positive test band was visible after 1–2 min at room temperature. - Analytical sensitivity: Limit of detection was approximately 2 × 10^2 ASFV genome copies, similar to qPCR sensitivity. Using dual crRNAs (crRNA1 + crRNA5) did not lower the LOD beyond 200 copies. - Field sample concordance: In 30 veterinary samples, Cas-gold results matched qPCR: all negatives showed only control bands; all positives showed both control and test bands. Example qPCR-quantified positives included 6.84E+02, 2.97E+02, 6.90E+03, and 1.96E+03 copies/μl, all detected by Cas-gold.

The study demonstrates that a CRISPR-Cas12a-based detection platform, coupled with isothermal amplification and a gold nanoparticle lateral flow readout, can achieve rapid, instrument-free ASFV detection with specificity comparable to laboratory-based qPCR. Careful crRNA selection against conserved regions of essential ASFV genes (DNA Pol, pp220) enabled broad subtype coverage while preserving specificity; mismatch analyses confirm stringent PAM and PAM-proximal requirements, minimizing false positives from unrelated targets. The optimized lateral flow configuration (reporter concentration, Cas12a/crRNA levels, brief reaction times at room temperature) facilitates practical use in field settings without specialized equipment. Consistent results across 30 veterinary samples and an LOD of ~200 copies underscore its potential utility for real-time surveillance and outbreak control, offering a sensitive, fast, and cost-effective alternative to PCR-dependent diagnostics.

This work presents a portable, rapid Cas12a-based paper diagnostic (Cas-gold) for ASFV that matches qPCR sensitivity (~200 copies) while eliminating instrument requirements. By identifying robust crRNAs to conserved ASFV targets and integrating RPA with an optimized lateral flow readout, the assay delivers accurate detection from swine blood and swabs within minutes at room temperature, with high specificity and no cross-reactivity to common swine pathogens. Future efforts could expand validation across more diverse ASFV strains and field conditions, refine crRNA panels for emerging variants, streamline sample preparation, and adapt the platform to multiplex detection of other swine pathogens.

- Partial tolerance to certain single-nucleotide mutations at PAM-distal positions could affect detection of variant strains or produce variable signal strength. - Initial lateral flow conditions produced false-positive test bands in blanks until reporter concentration was optimized, indicating susceptibility to assay configuration. - Fluorescence validation required PCR pre-amplification; while the lateral-flow workflow used RPA, broader field validation across sample types and environmental conditions is not detailed here. - The full set of author affiliations indicates additional institutions not fully listed in the excerpt; broader multi-center clinical validation data are not presented. - Although specificity was tested against several swine pathogens (and JEV in lateral flow), comprehensive cross-reactivity testing across a larger pathogen panel was limited in scope.