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.

Development and application of a multiplex PCR method for the simultaneous detection and differentiation of feline panleukopenia virus, feline bocavirus, and feline astrovirus

“…the application of routine PCR for the simultaneous detection and differentiation of these three viruses. Multiplex PCR (mPCR) can simultaneously detect and distinguish two or more target viruses in one reaction, can save time and cost, and has been widely used in large-scale epidemiological investigations [8,28]. In the present study, a mPCR assay was developed by combining three pairs of universal primers targeting different members of the species Carnivore protoparvovirus 1, FBoV and FeAstV in one reaction.…”

Porcine Bocavirus: A 10-Year History since Its Discovery

“…Conventional multiplex PCR can be used to detect the three groups of PBoV. The sensitivity of single PCR assays is equal to that of the multiplex PCR assay and is 1.0 9 10 3 genomic copies/lL for PBoV group 1, 4.5 9 10 3 copies/lL for PBoV group 2 and 3.8 9 10 3 copies/lL for PBoV group 3 (Zheng et al 2016a). Loopmediated isothermal amplification (LAMP) was developed for rapid, specific, and sensitive detection of porcine bocalike virus.…”

A novel porcine kobuvirus emerged in piglets with severe diarrhoea in China

“…To confirm the cause of the diarrhoea, eight commercial kits were used to test the presence of porcine common diarrhoea‐associated pathogens (including porcine circovirus type 2, pseudorabies virus, classical swine fever virus, porcine epidemic diarrhoea virus, porcine transmissible gastroenteritis virus, porcine rotavirus, pathogenic Escherichia coli and Salmonella ). In addition, four novel porcine diarrhoea‐related viruses (including PKV, porcine astrovirus, porcine deltacoronavirus and porcine bocavirus) were further tested according to previously published PCR protocols (Zhai et al., ; Zheng et al., ; Zhou et al., ).…”