Development and validation of a dual-fluorescent isothermal enzymatic recombinase amplification assay for the rapid differentiation of porcine reproductive and respiratory syndrome virus-1 and porcine reproductive and respiratory syndrome virus-2.

内容摘要

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen causing substantial economic losses to the global swine industry. The two genotypes, PRRSV-1 (European type) and PRRSV-2 (North American type), differ markedly in genetic characteristics and epidemiological patterns. The co-circulation and co-infection of these genotypes are increasingly reported. Concurrently, viral recombination events continue to shape PRRSV evolution. These scenarios collectively drive the need for rapid and accurate differential diagnostics. We aimed to establish and systematically optimize a dual-fluorescent enzymatic recombinase amplification (ERA) assay for simultaneous PRRSV-1 and PRRSV-2 identification. Highly conserved regions within the ORF7 gene of both genotypes were selected as targets, and genotype-specific primers and differentially labeled fluorescent probes were designed and screened. Reaction temperature, primer volume ratio, and probe volume ratio were optimized using an orthogonal experimental design, enabling efficient and synchronous dual-target amplification under isothermal conditions (42 °C). The developed assay exhibited high analytical specificity for PRRSV-1 and PRRSV-2. The limits of detection for PRRSV-1 and PRRSV-2 were 102 and 10 copies/μL, respectively. Clinical evaluation revealed an overall agreement rate of 98.78%. The developed dual-fluorescent ERA assay offered rapid reaction kinetics, simple operation, and high sensitivity and specificity. It enabled reliable differentiation of PRRSV-1 and PRRSV-2 without complex instrumentation, providing a practical and efficient tool for on-site diagnosis, epidemiological surveillance, and precise control of PRRS.