A triplex real-time PCR for differential detection of classical, variant and Bartha-K61 vaccine strains of pseudorabies virus

Pseudorabies (PR), also known as Aujeszky’s disease, is an economically important infectious disease of pigs and other animals caused by pseudorabies virus (PRV). Since late 2011, increasing numbers of PR outbreaks have been reported on many Bartha-K61-vaccinated pig farms in China, and emerging PRV variants that differ from classical PRV strains genetically and antigenically have been confirmed to be responsible for the outbreaks. Accordingly, there is a need to differentiate diverse PRV strains co-circulating in the field. Here, we developed and evaluated a triplex real-time PCR for differential detection of wild-type PRV (classical and variant strains) and gE/gI gene-deleted vaccine strains based on three differently labeled TaqMan probes. The detection limits of the assay were 0.5 TCID₅₀ for classical strains, 0.2 TCID₅₀ for variant strains and 0.05 TCID₅₀ for vaccine strains. The sensitivity was also determined to be 50, 50 and 5 copies for the TJ, SC and Bartha-K61 strain, respectively. The assay did not show cross-reactivity with several common porcine viruses. Reproducibility tests showed that the inter- and intra-assay coefficients of variation were less than 3 %. When testing a total of 234 clinical swine samples, the agreement between the triplex real-time PCR and virus isolation was 100 % (234/234) for classical strains, 99.5 % (233/234) for variant strains, and 100 % (234/234) for the Bartha-K61 vaccine strain. The results demonstrate that this method is sensitive and specific and will be useful for rapid detection and differentiation of diverse PRV strains.