Development of a TaqMan probe-based dual real time PCR assay for the identification of NADC34-like PRRSV

Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that induces reproductive disorders in sows and respiratory diseases in growing pigs. Recently, the NADC34-like strain of PRRSV has become more prevalent, with outbreaks occurring across pig farms in China. However, a reliable diagnostic method for the clinical detection of this strain has been absent. This study developed a TaqMan probe-based dual real-time quantitative PCR assay targeting the M and GP5 genes to specifically identify the NADC34-like PRRSV strain. The assay exhibited high specificity, detecting exclusively the NADC34-like strain without cross-reactivity with other PRRSV strains. The detection limits for pMD-M and pMD-GP5 plasmids were 2.67 × 10² and 1.35 × 10¹ copies/μL, respectively, indicating high assay sensitivity. The assay also demonstrated excellent reproducibility, with coefficient of variation (CV) values for both recombinant plasmids below 2 %. Among 251 clinical samples, 27 tested positive for NADC34-like PRRSV. This study establishes an accurate, sensitive, and reliable TaqMan dual real-time PCR assay for detecting NADC34-like PRRSV, offering a valuable tool for clinical diagnostics and outbreak management in pig farms.