ARS | Publication request: Validation of a real-time PCR assay for pseudorabies virus surveillance purposes

Submitted to: Conference Research Workers Disease Meeting
Publication Type: Abstract Only
Publication Acceptance Date: October 24, 2011
Publication Date: December 4, 2011
Citation: Miller, L.C., Zanella, E.L., Lager, K.M., Bigelow, T.T. 2011. Evaluation of a real-time PCR assay for pseudorabies virus surveillance purposes. Conference of Research Workers in Diseases [abstract]. Paper No. 062P.

Technical Abstract: Pseudorabies virus (PRV) is of significant economic importance for the swine industry worldwide. PRV is the cause of Aujeszky’s disease, also known as pseudorabies. The main symptoms are related to respiratory and nervous systems, which are the preferred site for PRV. Also, PRV can cause a high mortality in neonatal piglets, abortion in sows, and loss of body condition in the growing pigs. The feral swine population is known to be infected with PRV strains, and is considered to be a threat for the commercial swine industry. Real-time polymerase chain reaction (real-time PCR) is a valuable diagnostic technique that can rapidly identify nucleic acid of infectious agents in clinical specimens. A real-time PCR assay was designed based on the gB and gE genes to identify PRV nucleic acid in diagnostic samples. Using virus isolation as the gold standard test, the assay performed well in a variety of diagnostic matrices. Initial validation testing was conducted on 711 nasal swabs (sensitivity: 97.1% [95% confidence interval (confidence interval, CI): 95.8–98.8%], specificity: 78.6% [95% CI: 71.2–86.1%]); 258 brain tissues (sensitivity: 100.0% [95% CI: 100.0%], specificity: 92.1% [95% CI: 88.7–95.5%]); 132 bronchial-alveolar lung fluid specimens (sensitivity: 100.0% [95% CI: 100%], specificity: 76.9% [95% CI: 69.3–84.6%]); 29 preputial swabs fluid samples (sensitivity: 100.0% [95% CI: 100%], specificity: 88.5.5% [95% CI: 76.2–100%]); and 144 tonsil samples (sensitivity: 52.5% [95% CI: 37.0–68.0%], specificity: 93.3% [95% CI: 88.5–98.1%]). Low numbers of other sample matrices showed good agreement between results of virus isolation and PCR. Diagnostic performance of the real-time PCR assay developed as a testing method indicates that it is a rapid, accurate assay that is adaptable to a variety of PCR platforms and can provide reliable results on an array of clinical samples.