title: Development and validation of a multiplex conventional PCR assay for simultaneous detection and grouping of porcine bocaviruses.
abstract: Porcine bocavirus (PBoV), a newly described porcine parvovirus, has received attention because it can be commonly identified in clinically affected pigs including pigs with post-weaning multisystemic wasting syndrome (PWMS) and pigs with diarrhea. In recent years, novel PBoVs have been identified and were classified into three genogroups, but the ability to detect and classify these novel PBoVs is not comprehensive to date. In this study, a multiplex conventional PCR assay for simultaneous detection and grouping of PBoVs was developed by screening combinations of mixed primer pairs followed by optimization of the PCR conditions. This method exclusively amplifies targeted fragments of 531bp from the VP1 gene of PBoV G1, 291bp from the NP1 gene of PBoV G2, and 384bp from the NP1/VP1 gene of PBoV G3. The assay has a detection limit of 1.0×10(3)copies/μL for PBoV G1 4.5×10(3) for PBoV G2 and 3.8×10(3) for PBoV G3 based on testing mixed purified plasmid constructs containing the specific viral target fragments. The performance of the multiplex PCR assay was comparable to that of the single PCRs which used the same primer pairs. Using the newly established multiplex PCR assay, 227 field samples including faeces, serum and tissue samples from pigs were investigated. All three PBoV genogroups were detected in the clinical samples with a detection rate of 1.3%, 2.6% and 12.3%, respectively for PBoV G1, G2 and G3. Additionally, coinfections with two or more PBoV were detected in 1.7% of the samples investigated. These results indicate the multiplex PCR assay is specific, sensitive and rapid, and can be used for the detection and differentiation of single and multiple infections of the three PBoV genogroups 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; D000914:Antibodies, Viral / Q000097:blood*; D002645:Chickens; D004372:Ducks; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005585:Influenza in Birds / Q000175:diagnosis*; D012372:ROC Curve; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012698:Serologic Tests / Q000379:methods*; D014421:Turkey
chemical_list: D000914:Antibodies, Viral
keywords: Detection; Grouping; Multiplex PCR; Porcine bocaviruses; Prevalence rate