title: Rapid detection and grouping of porcine bocaviruses by an EvaGreen(®) based multiplex real-time PCR assay using melting curve analysis.
abstract: Several novel porcine bocaviruses (PBoVs) have been identified in pigs in recent years and association of these viruses with respiratory signs or diarrhea has been suggested. In this study, an EvaGreen(®)-based multiplex real-time PCR (EG-mPCR) with melting curve analysis was developed for simultaneous detection and grouping of novel PBoVs into the same genogroups G1, G2 and G3. Each target produced a specific amplicon with a melting peak of 81.3 ± 0.34 °C for PBoV G1, 78.2 ± 0.37 °C for PBoV G2, and 85.0 ± 0.29 °C for PBoV G3. Non-specific reactions were not observed when other pig viruses were used to assess the EG-mPCR assay. The sensitivity of the EG-mPCR assay using purified plasmid constructs containing the specific viral target fragments was 100 copies for PBoV G1, 50 for PBoV G2 and 100 for PBoV G3. The assay is able to detect and distinguish three PBoV groups with intra-assay and inter-assay variations ranging from 0.13 to 1.59%. The newly established EG-mPCR assay was validated with 227 field samples from pigs. PBoV G1, G2 and G3 was detected in 15.0%, 25.1% and 41.9% of the investigated samples and coinfections of two or three PBoV groups were also detected in 25.1% of the cases, indicating that all PBoV groups are prevalent in Chinese pigs. The agreement of the EG-mPCR assay with an EvaGreen-based singleplex real-time PCR (EG-sPCR) assay was 99.1%. This EG-mPCR will serve as a rapid, sensitive, reliable and cost effective alternative for routine surveillance testing of multiple PBoVs in pigs and will enhance our understanding of the epidemiological features and possible also pathogenetic changes associated with these viruses in pigs.
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: Gaopeng
authors.initials: G
authors.lastname: Liu
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: Zining
authors.initials: Z
authors.lastname: Wang
authors.affiliation: College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.
authors.forename: Zongqi
authors.initials: Z
authors.lastname: Yang
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
mesh_terms: D000818:Animals; D001483:Base Sequence; D052660:Bocavirus / Q000235:genetics* / Q000302:isolation & purification*; D017931:DNA Primers / Q000378:metabolism; D009691:Nucleic Acid Denaturation; D010802:Phylogeny; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D034421:Sus scrofa / Q000821:virology*
chemical_list: D017931:DNA Primers
keywords: Detection; EvaGreen; Genogroup; Melting curve; Multiplex real-time PCR; Porcine bocaviruses