Development, Evaluation, and Clinical Application of PRRSV-2 Vaccine-like Real-Time RT-PCR Assays

Abstract

In this study, we developed and validated (1) singleplex real-time RT-PCR assays for specific detection of five PRRSV-2 MLV vaccine viruses (Ingelvac MLV, Ingelvac ATP, Fostera, Prime Pac, and Prevacent) and (2) a four-plex real-time RT-PCR assay (IngelvacMLV/Fostera/Prevacent/XIPC) including the internal positive control XIPC for detecting and distinguishing the three most commonly used vaccines in the USA (Prevacent, Ingelvac MLV, and Fostera). The singleplex and 4-plex vaccine-like PCRs and the reference PCR (VetMAX^TM PRRSV NA&EU, Thermo Fisher Scientific, Waltham, MA, USA) did not cross-react with non-PRRSV swine viral and bacterial pathogens. The limits of detection of vaccine-like PCRs ranged from 25 to 50 genomic copies/reactions. The vaccine-like PCRs all had excellent intra-assay and inter-assay repeatability. Based on the testing of 531 clinical samples and in comparison to the reference PCR, the diagnostic sensitivity, specificity, and agreement were in the respective range of 94.67-100%, 100%, and 97.78-100% for singleplex PCRs and 94.94-100%, 100%, and 97.78-100% for the 4-plex PCR, with a C_T cutoff of 37. In addition, 45 PRRSV-2 isolates representing different genetic lineages/sublineages were tested with the vaccine-like PCRs and the results were verified with sequencing. In summary, the vaccine-like PCRs specifically detect the respective vaccine-like viruses with comparable performances to the reference PCR, and the 4-plex PCR allows to simultaneously detect and differentiate the three most commonly used vaccine viruses in the same sample. PRRSV-2 vaccine-like PCRs provide an additional tool for detecting and characterizing PRRSV-2.

Keywords: PRRSV; multiplex; porcine reproductive and respiratory syndrome virus; real-time RT-PCR; recombination; singleplex; vaccine-like.

Figure 1

Different mixtures of Ingelvac MLV, Fostera, and Prevacent viruses tested with the IngelvacMLV/Fostera/Prevacent/XIPC 4-plex PCR. The identities of the samples #1–#8 are denoted at the top. Amplification plots of the 8 samples tested with IngelvacMLV/Fostera/Prevacent/XIPC 4-plex PCR are shown at the bottom. The reporter dyes ABY, VIC, FAM, and Cy5 are shown in blue, black, green, and red color, respectively.