title: A sensitive multiplex real-time PCR panel for rapid diagnosis of viruses associated with porcine respiratory and reproductive disorders.
abstract: The objective of this study was to develop a multiplex real-time PCR panel using TaqMan probes for the detection and differentiation of porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus North American type (PRRSV-NA), pseudorabies virus (PRV), classical swine fever virus (CSFV), porcine parvovirus type 1 (PPV1) and Japanese encephalitis virus (JEV). Specific primer and probe combinations for PCV2, PRRSV, PRV, CSFV, PPV1 and JEV were selected within the conserved region of each viral genome. The multiplex real-time PCR panel which was run in two separate tubes was capable of specific detection of the six selected pig viruses, without cross-reactions with other non-targeted pig viruses. The detection limit of the assays was 10 copies/μL for PCV2, PRV, CSFV and PRRSV and 100 copies/μL for PPV and JEV. The two-tube multiplex real-time PCR panel showed 99.2% concordance with conventional PCR assays on 118 field samples. Overall, the multiplex real-time PCR panel provides a fast, specific, and sensitive diagnostic tool for detection of multiple viral pathogens in pigs and will be useful not only for diagnostics, or ecological, epidemiological and pathogenesis studies, but also for investigating host/virus or virus/virus interactions, particularly during coinfections.
authors.affiliation: College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.
authors.forename: Haigang
authors.initials: H
authors.lastname: Wu
authors.affiliation: College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.
authors.forename: Pinbin
authors.initials: P
authors.lastname: Rao
authors.affiliation: College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China. Electronic address: yonghoujiang@163.com.
authors.forename: Yonghou
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: The Roslin Institute and The Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian EH25 9RG, UK; Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, USA.
authors.forename: Tanja
authors.initials: T
authors.lastname: Opriessnig
authors.affiliation: College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.
authors.forename: Zongqi
authors.initials: Z
authors.lastname: Yang
mesh_terms: D000818:Animals; D054884:Host-Pathogen Interactions; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D019318:Porcine Reproductive and Respiratory Syndrome / Q000175:diagnosis* / Q000821:virology; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics / Q000502:physiology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000821:virology; D014780:Viruses / Q000145:classification / Q000235:genetics*
keywords: Multiplex real-time PCR panel; Porcine viruses; Respiratory and reproductive disorders; TaqMan probe