Development and analytical validation of a quantitative PCR assay for the detection of Magellanic penguin herpesvirus 1

Abstract

Herpesviruses are associated with disease in several aquatic bird species, including penguins. Magellanic penguin herpesvirus 1 (MagHV1) was initially detected in 58.3 % of oiled Magellanic penguins (Spheniscus magellanicus) in South America presenting with respiratory distress characterized by a combination of necrohemorrhagic tracheitis, fibrinous air sacculitis, pneumonia, and death. Additional exploration is needed to understand how herpesviruses affect penguin health; however, there is currently a lack of rapid, sensitive, and specific methods for detecting and quantifying herpesvirus infections in this taxon. To address this problem, we developed a real-time quantitative PCR (qPCR) assay for the detection of MagHV1 in penguins. Using a commercial program, TaqMan-MGB primer-probes targeting the DNA polymerase gene were designed in silico. Inter- and intra-assay variability, dynamic range, limit of detection, and analytical specificity were assessed to validate the assay per MIQE guidelines. The resulting assay was highly specific for MagHV1, failing to amplify fifteen closely related avian herpesviruses. It performed with high efficiency (slope =-3.336, R² = 0.999, efficiency 99.40 %) and low inter- and intra-assay variability (coefficient of variation < 1.67 % at all dilutions). Reaction efficiency was not impacted by the presence of penguin DNA from known-negative tracheal swabs. This qPCR assay has a linear range of detection from 10⁷ to 10¹ viral copies per reaction and provides a valuable tool in the surveillance and characterization of MagHV1 epidemiology in penguins. This assay can further be used to detect asymptomatic birds and as an effective tool to monitor infectious individuals.