The porcine circovirus type 3 (PCV3) infection is an emerging disease associated with clinical signs of porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs. Currently, there is a lack of effective vaccines and therapeutics against this disease. Therefore, rapid, effective, sensitive, and specific detection methods are crucial for the timely identification, prevention, and control of PCV3. In this study, we developed one- and two-pot visual detection methods for PCV3 using a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a detection system combined with recombinase polymerase amplification (RPA). These two methods demonstrated no cross-reactivity with eight other swine viruses and exhibited minimum detection limits of five and two copies of viral DNA, respectively, revealing their high specificity and sensitivity. During a clinical sample detection within 30 min, the coincidence rates between the one- and two-pot detection methods and real-time quantitative polymerase chain reaction (qPCR) were 100%. In conclusion, both one- and two-pot RPA-CRISPR/Cas12a detection methods have significant potential for the rapid, sensitive, and specific visual detection of PCV3.

Senecavirus A (SVA) is an emerging pathogen responsible for vesicular lesions and neonatal mortality in swine. In the absence of effective vaccines or therapeutics, early and accurate diagnosis is essential for controlling SVA outbreaks. Although nucleic acid-based detection methods are commonly employed, there remains a pressing need for rapid, convenient, highly sensitive, and specific diagnostic tools. Here, we developed a two-pot assay combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a containing crRNA targeting canonical protospacer adjacent motifs (PAMs) for simple, rapid, and visual identification of SVA in clinical samples. Subsequently, we successfully streamlined this system into a one-pot assay by selecting a specially designed crRNA targeting suboptimal PAM and integrating RPA amplification reagents and CRISPR/Cas12a detection components into a single reaction system in one tube. The developed methods exhibited diagnostic specificity, showing no cross-reactivity with four major swine viruses, while showing remarkable sensitivity with a lower detection limit of just two copies. Clinical validation in field samples using these two methods revealed perfect agreement (100% concordance) with conventional quantitative PCR (qPCR) results (sample size, n = 28), with both assays completing detection within 30 min. These results demonstrate that both the one-pot and two-pot RPA-CRISPR/Cas12a assays offer a reliable and efficient method for detecting SVA in this pilot study. Despite the limited sample size, the assays combine rapid reaction time with high sensitivity and specificity, showing great potential for future diagnostic applications.