Extraction-free, rapid LAMP-CRISPR/Cas12a assay for detection of pseudorabies virus.

Abstract

This study developed a LAMP-CRISPR/Cas12a detection system for rapid and visual identification of porcine pseudorabies virus (PRV). Optimal sgRNA and LAMP-specific primers were designed based on the conserved sequences of the viral pathogenic gene gG. The combined detection system demonstrated superior sensitivity compared to PCR-CRISPR/Cas12a and qPCR methods, achieving a detection limit of 1.0 × 10⁻⁴ copies/μL for the target plasmid DNA. Specificity testing confirmed the selective identification of PRV without cross-reactivity to other porcine pathogens. Parallel comparison of 26 serum samples between LAMP-CRISPR/Cas12a and PCR-CRISPR/Cas12a systems showed 100% concordance for positive results, with both detecting 12 positive samples. The method eliminates the need for viral nucleic acid extraction and requires only a constant temperature device and/or basic fluorescence detection equipment. Results are obtainable within one hour and are readable by the naked eye. This simple, sensitive, and equipment-independent approach is ideal for on-site rapid diagnosis of pseudorabies in pigs, offering significant applications in clinical diagnosis, epidemiological surveillance, and field testing.