Differential diagnosis of PRRS infection and vaccination by one-step real-time RT-PCR

摘要

PRRS elimination projects monitor progress by reduction and disappearance of virulent PRRS V by diagnostic RT-PCR monitoring of serum or oral fluids. Elimination projects may use mass vaccination to homogenize immunity and displace field viruses at the onset of elimination projects and as an intervention strategy to reduce field virus pressure. However, vaccine use complicates diagnostic interpretation since positive results may be due to vaccine or field virus, such that extra costs and time are incurred to sequence positives for a definitive diagnosis. Sequencing requires more RNA than can be detected by RT-PCR, a critical limitation, especially in late stages of an elimination program, such as herd closure, in which weaned pig testing is approaching negative, when RT-PCR levels are near the level of detection, and thus below the sensitivity of sequencing. We developed one-step TaqMan RT-PCR assays for identification of PRRS V vaccine isolates in PRRSV-positive diagnostic samples. Primers and probes were designed to target the major envelope gene (ORF5) of reference vaccine strains Ingelvac PRRS MLV and In-gelvac PRRS ATP. Based on the optimized primer-probe sets to detect ORF5, the one-step real-time RT-PCR assay discriminate natural infectionfrom Ingelvac PRRS MLV or Ingelvac PRRS ATP. Thirty of762 serum samples positive by type 2 diagnostic PRRSV PCR were identified as Ingelvac PRRS MLV and 23 were identified as Ingelvac PRRS ATP. The data indicate that one-step TaqMan RT-PCR assays are a rapid and efficient method for large-scale screening for differentiation of infected from vaccinated animals (DIVA) in monitoring programs associated with PRRS control and elimination projects.