[ Objective ] To establish a real-time fluorescent quantitative polymerase chain reaction （PCR） method with SYBR Green I for the detection of porcine circovirus type 2 （PCV2）. [Methods] Specific primers were desig...[ Objective ] To establish a real-time fluorescent quantitative polymerase chain reaction （PCR） method with SYBR Green I for the detection of porcine circovirus type 2 （PCV2）. [Methods] Specific primers were designed to amplify the conserved gene segments of PCV2 with a size of 177 bp by PCR. The ampli- fied gene was cloned into the vector of pMD 18-T and transformed into DHSct to screen positive clones. After being extracted and purified, the recombinant plasraids pMD 18-T-177 were taken as the standard DNA templates to establish the fluorescence quantitative PCR method for the detection of PCV2, and the PCR re- action conditions were optimized. [ Results] Ct value of the established PCR method showed a good linear relationship with the standard DNA templates within a viral load of 3.21 × 100 -4.16 × 108 copies/μL , the correlation coefficient was O. 998 8 and the slope was - 3.286. The method did not show any cress-reactions with the genomes of PRRSV, PCV1, CSFV, PRV, PPV and Escherichia coli. Sensitivity of this method was proved to be 3.21 × 10 copies/μL, which was 1 000 times higher as conventional PCR method. Variation coefficients of the repeated trims among same batch or different batches were both less than 3.00%. Positive rate of clinical samples detected by the established PCR method was 58.94%, which was significantly higher than the detection rate by conventional PCR. [ Conclusions ] A reM-time fluorescent quantitative PCR method with SYBR Green I for the detection of PCV2 was established, which was better for conducting the quan- titative analysis and the early diagnosis of PCV2 infection.展开更多
ORF2 gene fragment was amplified from Porcine circovirus type 2（ PCV2） Shandong isolates by PCR,homology analysis and genotype identification were conducted on PCV2 ORF2 gene combining with the sequences listed in G...ORF2 gene fragment was amplified from Porcine circovirus type 2（ PCV2） Shandong isolates by PCR,homology analysis and genotype identification were conducted on PCV2 ORF2 gene combining with the sequences listed in Gen Bank. The dominating popular genotype of PCV2 in Shandong Province was developed and the gene genetic variation characteristics of PCV2 was discussed. Specific PCR primers were designed and synthesized according to the differences among PCV2 genetypes,PCV2 gene identification PCR detection was established,and feasibility of the method was evaluated from the respects of sensitivity,specificity and repeatability,etc A total of 28 PCV2 ORF2 complete genes were obtained including 4 PCV2 a isolates,21 PCV2 b isolates and 3 PCV2 d isolates,indicating that the dominating popular genotype of PCV2 in Shandong Province was PCV2 b. Analysis on gene homology showed that homology of isolates in different regions from2000 to 2012 was 90. 5%- 99. 8%,amino acid sites sequences existed in different genotypes of PCV2 were different. Fragments amplified by the established PCR method were 341 bp and 177 bp,the lowest content of DNA could be detected was 5 ng / μL. Specific test results showed that the DNA amplifications on PCV2 a and PCV2 b were both positive,while amplifications on Porcine circovirus virus 1（ PCV1）,Porcine reproductive and respiratory syndrome virus（ PRRSV）,Pseudorabies virus（ PRV）,Porcine parvovirus（ PPV）,Classical swine fever virus（ CSFV）,Japanese encephalitis virus（ JEV） and Escherichia coli（ E. coli） were all negative,all the tests were conducted for 10 times,and the results were consistent. Test results indicated that the established PCR method had good specificity and repeatability,which could be applied to the identification of PCV2 genotypes.展开更多
In order to obtain induction expressed porcine circovirus type 2（PCV2）multi-epitope with good immunogenicity in vitro.Major epitopes of PCV2 were screened in the test,epitopes were composed sequentially,the c DNA se...In order to obtain induction expressed porcine circovirus type 2（PCV2）multi-epitope with good immunogenicity in vitro.Major epitopes of PCV2 were screened in the test,epitopes were composed sequentially,the c DNA sequences were artificially synthesized.Bam HⅠand SalⅠwere directionally cloned into prokaryotic expression vector PEGX-4T-1 multiple cloning site,then BL21 competent cells were transformed,positive clones were screened,IPTG inducible expression was conducted.Expression on target gene was analyzed by SDS-PAGE,fusion protein polypeptide was extracted and purified,immunocompetence of the expressed multi-epitope protein was identified by Westernblot.BALB/c mouse was immuned by fusion protein polypeptide,the antibody was determined by ELISA,immunogenicity was evaluated.Results showed that expression recombinant plasmid pEGX-4T-1-ep contained with seven PCV2 antigen epitopes had been constructed successfully.SDS-PAGE analysis showed that fusion protein polypeptide was expressed effectively in Escherichia coli（E.coli）,and the molecular weight was about 35ku,which existed in the form of solubility.Results of Westernblot showed that the extraction and purification of fusion protein polypeptide and PCV2 positive serum had good reactogenicity.Results of ELISA showed that the purified fusion protein polypeptide could stimulate the body to produce PCV2 specific antibody which had good immunogenicity.Results indicated that the constructed PCV2 multi-epitope had good expression characteristics in vitro,and the expression protein had good immunogenicity.The study provided a basic for the study on PCV2 epitope screening,functional identification and multi-epitope vaccine.展开更多
基金Supported by Shandong Province Natural Science Fund Project
文摘[ Objective ] To establish a real-time fluorescent quantitative polymerase chain reaction （PCR） method with SYBR Green I for the detection of porcine circovirus type 2 （PCV2）. [Methods] Specific primers were designed to amplify the conserved gene segments of PCV2 with a size of 177 bp by PCR. The ampli- fied gene was cloned into the vector of pMD 18-T and transformed into DHSct to screen positive clones. After being extracted and purified, the recombinant plasraids pMD 18-T-177 were taken as the standard DNA templates to establish the fluorescence quantitative PCR method for the detection of PCV2, and the PCR re- action conditions were optimized. [ Results] Ct value of the established PCR method showed a good linear relationship with the standard DNA templates within a viral load of 3.21 × 100 -4.16 × 108 copies/μL , the correlation coefficient was O. 998 8 and the slope was - 3.286. The method did not show any cress-reactions with the genomes of PRRSV, PCV1, CSFV, PRV, PPV and Escherichia coli. Sensitivity of this method was proved to be 3.21 × 10 copies/μL, which was 1 000 times higher as conventional PCR method. Variation coefficients of the repeated trims among same batch or different batches were both less than 3.00%. Positive rate of clinical samples detected by the established PCR method was 58.94%, which was significantly higher than the detection rate by conventional PCR. [ Conclusions ] A reM-time fluorescent quantitative PCR method with SYBR Green I for the detection of PCV2 was established, which was better for conducting the quan- titative analysis and the early diagnosis of PCV2 infection.
基金Supported by Shandong Province Natural Science Foundation of China(ZR2013CQ006)
文摘ORF2 gene fragment was amplified from Porcine circovirus type 2（ PCV2） Shandong isolates by PCR,homology analysis and genotype identification were conducted on PCV2 ORF2 gene combining with the sequences listed in Gen Bank. The dominating popular genotype of PCV2 in Shandong Province was developed and the gene genetic variation characteristics of PCV2 was discussed. Specific PCR primers were designed and synthesized according to the differences among PCV2 genetypes,PCV2 gene identification PCR detection was established,and feasibility of the method was evaluated from the respects of sensitivity,specificity and repeatability,etc A total of 28 PCV2 ORF2 complete genes were obtained including 4 PCV2 a isolates,21 PCV2 b isolates and 3 PCV2 d isolates,indicating that the dominating popular genotype of PCV2 in Shandong Province was PCV2 b. Analysis on gene homology showed that homology of isolates in different regions from2000 to 2012 was 90. 5%- 99. 8%,amino acid sites sequences existed in different genotypes of PCV2 were different. Fragments amplified by the established PCR method were 341 bp and 177 bp,the lowest content of DNA could be detected was 5 ng / μL. Specific test results showed that the DNA amplifications on PCV2 a and PCV2 b were both positive,while amplifications on Porcine circovirus virus 1（ PCV1）,Porcine reproductive and respiratory syndrome virus（ PRRSV）,Pseudorabies virus（ PRV）,Porcine parvovirus（ PPV）,Classical swine fever virus（ CSFV）,Japanese encephalitis virus（ JEV） and Escherichia coli（ E. coli） were all negative,all the tests were conducted for 10 times,and the results were consistent. Test results indicated that the established PCR method had good specificity and repeatability,which could be applied to the identification of PCV2 genotypes.
基金Supported by Shandong Province Natural Science Foundation of China(ZR2013CQ006)
文摘In order to obtain induction expressed porcine circovirus type 2（PCV2）multi-epitope with good immunogenicity in vitro.Major epitopes of PCV2 were screened in the test,epitopes were composed sequentially,the c DNA sequences were artificially synthesized.Bam HⅠand SalⅠwere directionally cloned into prokaryotic expression vector PEGX-4T-1 multiple cloning site,then BL21 competent cells were transformed,positive clones were screened,IPTG inducible expression was conducted.Expression on target gene was analyzed by SDS-PAGE,fusion protein polypeptide was extracted and purified,immunocompetence of the expressed multi-epitope protein was identified by Westernblot.BALB/c mouse was immuned by fusion protein polypeptide,the antibody was determined by ELISA,immunogenicity was evaluated.Results showed that expression recombinant plasmid pEGX-4T-1-ep contained with seven PCV2 antigen epitopes had been constructed successfully.SDS-PAGE analysis showed that fusion protein polypeptide was expressed effectively in Escherichia coli（E.coli）,and the molecular weight was about 35ku,which existed in the form of solubility.Results of Westernblot showed that the extraction and purification of fusion protein polypeptide and PCV2 positive serum had good reactogenicity.Results of ELISA showed that the purified fusion protein polypeptide could stimulate the body to produce PCV2 specific antibody which had good immunogenicity.Results indicated that the constructed PCV2 multi-epitope had good expression characteristics in vitro,and the expression protein had good immunogenicity.The study provided a basic for the study on PCV2 epitope screening,functional identification and multi-epitope vaccine.
