[A rapid visual detection method for porcine circovirus type 4 based on enzymatic recombinase amplification and...

The emerging outbreaks of infectious diseases in humans and animals worldwide pose serious threats to public health and cause substantial economic losses. This has raised the demand for more efficient and sensitive diagnostic methods to strengthen disease surveillance and early warning. In this study, we developed a portable visual platform based on ERA-CRISPR/EsCas13d for the rapid detection of porcine circovirus type 4 under resource-limited conditions. We optimized the platform by integrating enzymatic recombinase amplification (ERA), T7 transcription, and CRISPR/EsCas13d cleavage in a single-tube reaction, thereby simplifying the workflow and shortening the total detection time to 30 min. In addition, a rapid nucleic acid release method was employed, eliminating the need for laboratory-based extraction and complex heating steps, which further improved the simplicity and usability. In addition, lyophilized reagents were modified to enhance stability, thus reducing cold-chain and storage requirements and enabling cost-effective transport and field deployment. The method achieved a visual limit of detection of 50 cp/μL for porcine circovirus type 4 and demonstrated no cross-reactivity with six other common swine pathogens. Moreover, the platform supported two visual readout formats-UV light (470 nm) and lateral flow assay (LFA)-providing flexible options for interpretation in different application scenarios. Validation with 60 clinical samples showed 100% concordance with quantitative PCR results, highlighting the practical utility and application potential of the ERA-CRISPR/EsCas13d-based portable visual platform for rapid on-site diagnostics.