Development of a one-step reverse transcription-quantitative polymerase chain reaction assay for the detection of porcine reproductive and respiratory syndrome virus

Abstract

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is an important disease that severely affects the swine industry and, therefore, warrants rapid and accurate diagnosis for its control. Despite the progress in developing diagnostic tools, including polymerase chain reaction (PCR)-based methods such as reverse transcription quantitative PCR (RT-qPCR) to diagnose PRRSV infection, its diagnosis at the genetic level is challenging because of its high genetic variability. Nevertheless, RT-qPCR is the easiest and fastest method for diagnosing PRRSV. Therefore, this study aimed to develop an RT-qPCR assay for rapid and accurate diagnosis of PRRSV by encompassing all publicly available PRRSV sequences. The developed assay using highly specific primers and probes could detect up to 10 copies of PRRSV-1 and -2 subtypes. Furthermore, a comparison of the performance of the developed assay with those of two commercial kits widely used in South Korea demonstrated the higher efficiency of the developed assay in detecting PRRSV infections in field samples. For PRRSV-1 detection, the developed assay showed a diagnostic agreement of 97.7% with the results of ORF5 sequencing, while for commercial kits, it showed 95.3% and 72.1% agreement. For PRRSV-2, the developed assay showed a diagnostic agreement of 97.7%, whereas the commercial kits showed 93% and 90.7% agreement. In conclusion, we developed an assay with higher accuracy than those of the tested commercial kits, which will contribute markedly to global PRRSV control.

Fig 1. The analytic sensitivity results and standard curve of the developed RT-qPCR.

The developed RT-qPCR’s amplification graphs and standard curves were generated using ten-fold dilutions of plasmid (1–10⁸ copies). (A) PRRSV-1 and (B) PRRSV-2.

Fig 2

The phylogenetic tree analysis of the field samples of PRRSV-1 (A) and PRRSV-2 (B) using ORF5 Sanger sequencing results.