The Cloning Of Porcine Bocavirus Nonstructural Gene And The Establishing And Application Of RT-PCR

Porcine Bocavirus belongs to the Family Parvoviridae,Genus Bocavirus of new members.But few messages about the pathogenicity of bocavirus had recognized clearly.The key reason for this is that there was lack of effective culture or porcine model,causing the studies for the replication and pathogenesis of bocavirus remains limited.Now there are many genotypes for porcine bocavirus,such as:PBoV1,PBoV2,PBoV3,PBoV4 and PBoV5.There are many countries in different continents had reported the piglets infected with bocavirus.Though there are a lot of PBoV complete sequences,available in GenBank,a little report about the sequence of Fujian samples.The study on Fujian samples is helpful for look at the PBoV biology information and establishing a rapid,effective detection methods.The research contents are as follow:1,Clone and sequence the NS1 gene for PBoV isolated in FujianIn this study,the NS1 gene of the porcine bocavirus isolates PBoV-FJ2014-01 strains was cloned by PCR methods with different specific primers with two parts.The results showed that the full-length of the NS1 gene is 2004 nucleotides,encoding667 amino acids.The PBoV isolate PBoV-FJ2014-01 shared the same phylogenetic relationship with the PBoV isolates reported preciously.The NS1 gene of PBoV isolate PBoV-FJ2014-01 was compared with other respective PBoV isolates.The results showed that the highest nucleotides homology with PBoV5 isolates 5/JS677(GenBank accession number JN831651)at 98.4%,also shared 96.0%with another PBoV5 isolates HB11(GenBank accession number JN621325).The results also showed that the PBoV-FJ2014-01 shared nucleotides homology with other PBoV isolates among 80.0%to 90.0%.However,the PBoV-FJ2014-01 shared nucleotides homology with PBoV1pig/ZJD/China/2006(GenBank accession number HM05369)and PBoV2pig/ZJD/China/2006(GenBank accession number HM053694)at 52.5%and 53.2%,derived the PBoV-FJ2014-01 belong to porcine bocavirus type 5.2,Establish the SYBR Green I real-time quantitative PCR method for PBoV 5 and used for epidemiological investigationIn order to develop more sensitive and specific diagnostic test for PBoV 5 by using the real time quantitative PCR technology based SYBR I.Comparing with other PBoV 5 isolates from the GenBank,a SYBR I based real time quantitative PCR methods was established,which can be used for PBoV 5 epidemiological investigation in Fujian,China.This method is linear with good relations(r is 0.999)and good amplification efficiency.The detection assay was linear in the range of 3.64×102?3.64×107 copies/?L.There was no cross-reaction occurred with nucleic acids extracted from porcine circovirus(type 1 and type 2),porcine parvovirus and porcine torque teno virus(genogroup 1 and genogroup 2).The intra-assay CVs were among 0.35%to 1.24%and also the inter-assay CVs among 0.43%to 1.46%.There was 7/74 with piglets feces,6/142 with sera samples and 11/94 with tissues collected in different cities in Fujian,China.In conclusion,this study obtained the full-length NS1 gene of PBoV5 isolates PBoV-FJ2014-01,and molecular characterization were analysis for the cloned sequences.A SYBR ? based real time quantitative PCR method was established for detecting the samples epidemiological investigation for PBoV5.