Establishment And Application Of A Fluorescent Quantitative Pcr For Porcine Bocavirus And Its Molecular Epidemiologyin Partial Regions Of China

In members of the Bocavirus genus, that contain three open reading frames (ORFs), of the Parvovirinae subfamily, porcine bocaviruses (PoBoVs) exhibit the most genetic diversity. Based on the ORF2-encoded viral protein (VP1) classification, the fifteen reported porcine bocaviruses were grouped into nine species:PoBoV1(porcine boca-like virus, PBo-likeV and PBoVI-H8), PoBoV2(porcine parvovirus4-cl4, PPV4-cl4and PPV4-cl7),PoBoV3(PBoV1, PBoV2and PBoV2-A6), PoBoV4(6V), PoBoV5(PBoV3-NI), PoBoV6(PBoV4-NI), PoBoV7(PBoV3-HK), PoBoV8(PBoV4-1-HK and PBoV4-2-HK) and PoBoV9(7V and PBoV5), with PoBoV3and PoBoV9both having two genotype viruses, respectively. PBo-likeV, PPV4, PBoV1, PBoV2,6V,7V, PBoV3-NI and PBoV4-NI were detected among the166samples collected in2010from swine herds located in ten provinces (city) of China. The detection rates were28.9%,6.6%,9.0%,12.7%,22.3%,31.3%,15.1%and14.5%, respectively. The co-infection combinations involving these eight porcine bocaviruses in the collected samples were very common. Furthermore, mixed-infections with viruses from other families (porcine reproductive and respiratory syndrome virus, classic swine fever virus and porcine circovirus type2) were also detected.The NP1gene of PoBoVl was amplified by PCR from plasmid and cloned into pET-32(P3) vector to construct a recombinant plasmid NP1-pET32(P3). The recombinant protein was expressed with IPTG induction after NP1-pET32(P3) was transformed into BL21(DE3), and the expressed protein was about55KD which mainly in soluble form in the bacteria. The NP1protein was purified by the Ni-NTA resin and immunized in rabbits to prepare antisera. Antibody titer of blood serum tested by ELISA was1:64000, identified by Western-blot showed the antisera could react with the NP1protein.According to the only nucleotide sequence of PoBoV1published in GenBank at that time(Accession number:FJ872544), based on its conservative VP1gene, a TaqMan probe and one pair of primers were designed and synthesized for the establishment of Taqman fluorescent quantitative PCR(qPCR). Recombinant plasmid pPBoVZJ0901constructed by our laboratory was used to generate standard curve. qPCR was established by optimizing the annealing temperature, the total reacting system, the probe concentration and the DNA templates concentration. The specificity test showed that the qPCR could not detect plasmid of the full length porcine reproductive and respiratory syndrome virus, cDNA of classical swine fever virus, DNA of porcine circovirus type2and other types of porcine bocavirus, except positive DNA of PoBoV1. The sensitivity test indicated that lowest detectable limit of the assay was4.89×100copies/μl, with a broad linear detection ranged from4.89×100copies/μl～4.89×109copies/μl. The reproductive test revealed that inter-assay and intra-assay coefficient of variation of this assay were both less than3%. Samples of sera collected in different date and tissues collected after dissection were detected by the established real-time PCR for virus content, the results demonstrated that the virus loading level of sera were very low, approximately4.89x101copies/μl. The virus content of kidney was the highest among all of the tissues (1.83×104copies/ul).