African swine fever (ASF), caused by the ASFV, is a devastating disease for swine populations worldwide, resulting in substantial economic losses and impacting food security. The lack of effective vaccines and treatments necessitates rapid and accurate diagnostic tools for effective disease control and surveillance. Current methods, such as virus isolation, fluorescent antibody tests (FAT), and PCR, have limitations in terms of speed, sensitivity, and field applicability. Virus isolation is the gold standard but is time-consuming. FAT lacks sensitivity. While PCR is sensitive and fast, it requires specialized equipment and trained personnel, limiting its use in field settings. Isothermal amplification techniques offer advantages in terms of simplicity, but concerns regarding nonspecificity and high false-positive rates persist. The CRISPR-Cas system presents an alternative, offering high specificity and sensitivity in nucleic acid detection. This study leverages the collateral cleavage activity of Cas12a, along with isothermal amplification and a paper-based lateral flow strip for visual readout, to develop a portable and affordable ASFV diagnostic tool.

Existing ASFV detection methods include virus isolation (gold standard but time-consuming), antigen measurement via FAT (lacks sensitivity), and PCR (requires equipment and expertise). Isothermal amplification methods offer speed and simplicity but suffer from nonspecificity and high false-positive rates. CRISPR-Cas systems have emerged as powerful tools for nucleic acid detection, with Cas12a's collateral cleavage activity particularly promising due to its simplicity and cost-effectiveness compared to Cas13a-based methods.

The researchers designed six crRNAs targeting conserved regions of ASFV's pp220 and DNA polymerase genes. In vitro cleavage assays assessed the specificity and efficiency of these crRNAs. The optimal crRNAs (crRNA1 and crRNA5) were selected based on fluorescence intensity and activity kinetics using a FAM-Quencher reporter. Mismatch mutation tolerance was evaluated by introducing mutations into the target DNA sequences. Specificity was tested against other common swine pathogens (PRV, PCV, SS, MH, HPS). For veterinary sample testing, DNA was extracted from blood and anal swabs from ASFV-infected and uninfected pigs. The Cas12a-based fluorescence detection was compared to qPCR. A paper-based lateral flow assay was developed, integrating recombinase polymerase amplification (RPA) for rapid amplification and a gold nanoparticle-antibody conjugate for visual detection. The optimal concentration of FAM-Biotin ssDNA reporter and LbCas12a/crRNA complex was determined. The detection limit was established using ASFV genome copies. The accuracy and specificity of the lateral flow assay were evaluated using veterinary samples and other swine pathogens.

The study identified two highly specific crRNAs (crRNA1 and crRNA5) that effectively targeted ASFV DNA with minimal cross-reactivity to other swine pathogens. The Cas12a-based fluorescence assay demonstrated high specificity, showing low tolerance for mismatch mutations and no cross-reactivity with other common swine pathogens. Using samples from ASFV-infected pigs, the Cas12a-based fluorescence detection showed consistent results with qPCR, confirming the reliability of the assay. A portable paper-based lateral flow assay was successfully developed, combining RPA and Cas12a detection. This assay achieved a detection limit of 200 copies of ASFV genome, comparable to the qPCR method. The lateral flow assay showed high accuracy and specificity when tested on veterinary samples, displaying a clear positive band for ASFV-positive samples and a control band only for negative samples.

This study successfully developed a highly sensitive, specific, and portable diagnostic tool for ASFV detection. The use of Cas12a and a paper-based lateral flow strip addresses the limitations of existing methods by providing a rapid, instrument-free, and cost-effective alternative. The comparable sensitivity to qPCR highlights the assay's potential for large-scale field applications. The ability to detect ASFV directly from clinical samples underscores its practicality for on-site diagnosis, facilitating timely disease control interventions. This technology holds significant promise for improving ASF surveillance and reducing economic losses associated with this devastating disease.

The development of the Cas12a-mediated paper-based lateral flow assay offers a significant advancement in ASFV diagnostics. Its ease of use, affordability, and high sensitivity make it a promising tool for on-site detection and disease control efforts. Future studies could focus on optimizing the assay for even broader applications, including adapting it for other viral diseases and exploring the potential of multiplexing for simultaneous detection of multiple pathogens.

While the assay showed high sensitivity and specificity, the study's sample size was limited. Further validation studies with larger sample sets are necessary to fully assess its performance in diverse settings. Potential environmental factors influencing assay performance were not explicitly addressed.