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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 531 bp from the VP1 gene of PBoV G1, 291 bp from the NP1 gene of PBoV G2, and 384 bp 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]