Detection And Grouping Of Porcine Bocaviruses By Multiplex PCR

Porcine bocavirus(PBoV)is a linear single-stranded DNA virus belonging to genus Bocaparvovirus.Since it was first discovered in Swedish pigs with post-weaning multisystemic wasting syndrome(PMWS)in 2009,PBoV has been detected globally.Many novel PBoVs have been identified and classified into three PBoV groups in pigs in recent years,however,the detection and typing of porcine bocaviruses methods are not comprehensive to date.It is therefore important to develop an effective and reliable approach to demonstrate multiple genotypes with one single assay.In this study,two sets of assays including a multiplex conventional PCR and a multiplex real-time PCR were developed for simultaneous detection and grouping of the three PBoV groups in China.In the current study,a multiplex PCR assay was first developed for simultaneous detection and grouping of porcine bocaviruses.This method is able to specifically detect and distinguish three PBoV groups with a detection limit of 1.0×103 PBoV G1,4.5×103 PBoV G2,and 3.8×103 PBoV G3 copies/?L.Using newly established multiplex PCR assays,227 samples from pigs were investigated.The detection rate of PBoV G1,PBoV G2 and PBoV G3 was 1.3%,2.6% and 12.3% respectively in the investigated samples and the infections were diverse and complex with some samples infected by two or more PBoV groups in the same sample.The detection result of PBoV by the multiplex PCR was the same to the single PCR except that one sample was negative by the multiplex PCR but positive by the single PCR.The multiplex real-time PCR assay was then developed based on the melting curve analysis of different amplicons of each PBoV group using an EvaGreen dye with a distinct melting temperature(Tm)for each specific melting peak.The Tm values of the three PBoV groups are 81.3 ± 0.34 °C,78.2 ± 0.37 °C and 85.0 ± 0.29 °C,respectively,which can easily be differentiated from each other and used for identification of each PBoV group.The assay did not generate any specific melting peak when non targeted pig viruses and blank control were tested,suggestive of good specificity.The detection limit of the multiplex real-time PCR for PBoV G1,PBoV G2 and PBoV G3 was 100,50,100 copies/?L respectively,which was 4-5 fold less sensitive than that of the single real-time PCR assay.When the three PBoV group templates were mixed to stimulate the natural situation,the detection limit of the multiplex real-time PCR was 250 copies/?L for PBoV G1,250 copies/?L PBoV G2 for and 100 copies/?L for PBoV G3.The multiplex assay is highly repeatable with both intra-assay and inter-assay variation within 5%.These data demonstrated a good specificity,sensitivity,repeatability of the multiple EvaGreen real-time PCR.The established EvaGreen multiplex real-time PCR was then applied to the detection of clinical samples from pigs.Among 227 clinical samples,15.0%,25.1% and 41.9% were detected positive for PBoV G1,PBoV G2 and PBoV G3 by the multiplex real-time PCR,which compared to the single real-time PCR assay.In addion,the mixed infection of PBoV G1 and PBoV G2,PBoV G1 and PBoV G3,PBoV G2 and PBoV G3,PBoV G1,PBoV G2 and PBoV G3 was detected in 3.1%?5.7%?13.2% and 3.1% of 227 samples,respectively.A total positive rate of 55% indicated PBoV was widespread in China.The positive samples identified by these two assays were cloned to plasmids and sequenced for genetic diversity analysis of PBoV.Identity comparision of the nucleotide sequences was made between the measured fragment sequences of the positive samples and complete or nearly complete genomes of PBoV published in NCBI respectively.The homology of nucleotides was relatively high in the range of 72.1-100% in the same PBoV group,while the nucleotide sequence homology was low in the range of 3.2-54.2% between different PBoV groups.These results indicated that the PBoV grouping method into three PBoV groups and the two multiplex PCR assays described here were feasible and representative.In summary,the two multiplex PCR assays described here provide tools for simultaneous and rapid detection of PBoV G1,PBoV G2 and PBoV G3 in swine for epidemiological surveillance and disease management.