Rapid and visual differentiation of pseudorabies virus vaccine and wild-type strains using an RPA-LFD assay

Pseudorabies virus (PRV) inflicts substantial economic losses on the global swine industry. Accurate differentiation between vaccine-derived and circulating wild-type strains is critical for effective disease control and eradication programs. However, conventional diagnostic methods often rely on costly instrumentation and entail lengthy turnaround times, thereby limiting their utility for rapid, field-deployable differential diagnosis—particularly in resource-constrained settings. In this study, we developed a rapid detection system integrating recombinase polymerase amplification (RPA) with lateral flow diagnostic strips (LFD) for the simultaneous identification of PRV wild-type and vaccine strains. The assay demonstrated high analytical specificity, reliably differentiating between PRV vaccine and wild-type strains, exhibiting no cross-reactivity with other prevalent swine pathogens. It also exhibited high sensitivity, yielding clear visual results within 20 min and achieving a limit of detection of 80 copies/µL for the PRV gE gene. Moreover, the method is operationally straightforward, requires minimal training, delivers unambiguous visual readouts, and is highly cost-effective. Collectively, the RPA-LFD platform represents a robust, field-adaptable tool for on-site surveillance of PRV field strains, offering significant potential to support large-scale screening and advance pseudorabies eradication initiatives.