Cloning Of Porcine Circovirus Type 3 Complete Genome And Establishment And Application Of Real-time PCR Method

Porcine circovirus type 3(PCV3)is a new type of porcine circovirus that was first discovered in 2016.Epidemiological investigations showed that PCV3 often infected pigs with symptoms of PDNS and reproductive disorders.since porcine circovirus type 3 is already prevalent in the world,rapid and accurate diagnosis of the infection status of pigs is very significance important for preventing and controlling the disease.In this paper,a PCV3 conserved gene fragment(152 bp)was amplified by PCR and cloned into pMD-19T vector to construct the recombinant plasmid pMD19T-PCV2-Cap as a positive standard plasmid(also as a standard plasmid for both methods).It performs sensitivity,specificity and reproducibility verification.The results are as follows:1SYBR Green I real-time PCR:The standard has a good linear relationship in the range of 4.5×1010?4.5×103 copies/?L,with a correlation coefficient of 0.998 and a slope of 3.448;strong specificity,and PRRSV,PCV2,PPV,JEV and PRV have no cross-reaction,the sensitivity is 4.5×10opies/?L,which is 10 times higher than the conventional PCR method,and the coefficient of variation between the groups and the intra-group repeatability test is less than 2%;using SYBR Green I PCR detection of 435 pig farm serum samples,the positive rate was 65.7%,higher than the normal PCR positive detection rate of 51.7%,indicating that the established PCV3 SYBR Green I PCR method is sensitive and specific,and can be used for rapid detection of PCV3 infection in pig farms.Due to the better specificity of TaqMan,this study established a quantitative PCR assay based on TaqMan probe and tested the samples.2.TaqMan real-time PCR:The standard showed a good linear relationship in the range of 4.5×1011?4.5×103 copies/?L,with a correlation coefficient of 1,a slope of 3.982;strong specificity,sensitivity of 4.5×102 copies/?L,The coefficient of variation of the repeat test between groups and groups was less than 2%;435 pig farm serum samples were detected by TaqMan real-time PCR,and the positive rate was 70.11%,which was higher than the normal PCR positive detection rate of 51.7%.The successful TaqMan real-time PCR method was established.3.Sequence analysis of 5 PCV3 whole genes:Some pig tissue samples from Jilin Province were detected by the fluorescence quantitative method established in this study,and the whole sequence was amplified.After sequencing and correct identification,5 PCV3 whole gene sequences were obtained.Through genetic evolution analysis and nucleotide homology analysis,the nucleotide homology between the five strains was 98,7%?99.5%,and the nucleotide homology with 20 domestic PCV3 sequences was 98.7%?100.%,the homology with 10 foreign PCV3 sequences was 98.4%-99.6%.The two fluorescence quantification methods established in this paper can be used for the detection and epidemiological investigation of PCV3,laying the foundation for the prevention and treatment of PCV3;amplifying the whole genome of PCV3 and analyzing it accordingly,which provides a basis for the research on the trend of PCV3.