title: Development of an EvaGreen-based multiplex real-time PCR assay with melting curve analysis for simultaneous detection and differentiation of six viral pathogens of porcine reproductive and respiratory disorder.
abstract: Concurrent infection of pigs with two or more pathogens is common in pigs under intensive rearing conditions. Porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), Japanese encephalitis virus (JEV) and pseudorabies virus (PRV) are all associated with reproductive or respiratory disorders or both and can cause significant economic losses in pig production worldwide. An EvaGreen-based multiplex real-time PCR (EG-mPCR) with melting curve analysis was developed in this study for simultaneous detection and differentiation of these six viruses in pigs. This method is able to detect and distinguish PCV2, PPV, PRRSV, CSFV, JEV and PRV with the limits of detection ranging from 100 to 500 copies/μL, high reproducibility, and intra-assay and inter-assay variation ranging from 0.11 to 3.20%. After validation, a total of 118 field samples were tested by the newly developed EG-mPCR. PCV2 was identified in 23%, PPV in 15%, PRRSV in 17% and PRV in 5% of the samples. Concurrent PCV2 and PRRSV infection was detected in 6.7%, PCV2 and PPV in 5% and PPV2 and PRRSV infection was detected in 5% of the cases. The agreement of the EG-mPCR and conventional PCR tests was 99.2%. This EG-mPCR will be a useful, rapid, reliable and cost-effective alternative for routine surveillance testing of viral infections in pigs.
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.
authors.forename: Haigang
authors.initials: H
authors.lastname: Wu
authors.affiliation: College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China. Electronic address: yonghoujiang@zstu.edu.cn.
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: Xiaowen
authors.initials: X
authors.lastname: Zheng
authors.affiliation: College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.
authors.forename: Yecheng
authors.initials: Y
authors.lastname: Mo
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; D003365:Costs and Cost Analysis; D004267:DNA Viruses / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D025202:Molecular Diagnostic Techniques / Q000191:economics / Q000379:methods; D060885:Multiplex Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012328:RNA Viruses / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D015203:Reproducibility of Results; D060737:Reproductive Tract Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D012141:Respiratory Tract Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000821:virology; D013997:Time Factors; D044366:Transition Temperature; D014730:Veterinary Medicine / Q000191:economics / Q000379:methods*; D014777:Virus Diseases / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology
keywords: EvaGreen; Melting curve; Multiplex real-time PCR; Pig viruses; Surveillance