According to VP2 gene sequence of the porcine parvovirus virus strain NADL-2 （NC001718） available in GenBank （NC_001718）, a pair of specific primer was designed, and the target fragment of 431 bp was obtained by P...According to VP2 gene sequence of the porcine parvovirus virus strain NADL-2 （NC001718） available in GenBank （NC_001718）, a pair of specific primer was designed, and the target fragment of 431 bp was obtained by PCR amplification. The products were ligated with pMD18- T vector and then transformed into bacteria DH5α for recombinant plasmid extraction. After PCR identification and sequencing, recombinant plasmid was used as a standard template to establish the standard curve of SYBR Green I fluorescence quantitative PCR. Sensitivity test, specificity test and repeatability test were also determined. The results indicated that there was a good linear relationship between threshold cycle of the standard curve and template concentration, R2 =0.997 6. Tm ranged from 82.3 to 82.9 ℃, while the sensitivity was 72.1 copies/μl with good specificity and repeatability. The developed SYBR Green I real-time quantitative PCR method to detect PPV VP2 gene laid the basis for further studies on patho- oenesis, early clinical diaonosis of this virus and quantitative analysis of PPV infection.展开更多
Three pairs of primer were designed for amplification of porcine pseudorabies virus （PRV） gB, gE, and TK gene by multiplex PCR （multi-PCR） in order to differentiate vaccine strains from field isolates. Three speci...Three pairs of primer were designed for amplification of porcine pseudorabies virus （PRV） gB, gE, and TK gene by multiplex PCR （multi-PCR） in order to differentiate vaccine strains from field isolates. Three specific bands were obtained respectively at the expected size, 427 bp （gB gene）, 298 bp （gEgene）, and 208 bp （TKgene）, Then four different gene-deleted vaccines of PRV were detected by multi-PCR. One ex- pected specific band was observed in one of samples, while two bands in the others. As shown by the detection results, the multi-PCR has high sensitivity and specificity and should be applied in pathogen diagnosis and epidemiological investigation in the future.展开更多
文摘According to VP2 gene sequence of the porcine parvovirus virus strain NADL-2 （NC001718） available in GenBank （NC_001718）, a pair of specific primer was designed, and the target fragment of 431 bp was obtained by PCR amplification. The products were ligated with pMD18- T vector and then transformed into bacteria DH5α for recombinant plasmid extraction. After PCR identification and sequencing, recombinant plasmid was used as a standard template to establish the standard curve of SYBR Green I fluorescence quantitative PCR. Sensitivity test, specificity test and repeatability test were also determined. The results indicated that there was a good linear relationship between threshold cycle of the standard curve and template concentration, R2 =0.997 6. Tm ranged from 82.3 to 82.9 ℃, while the sensitivity was 72.1 copies/μl with good specificity and repeatability. The developed SYBR Green I real-time quantitative PCR method to detect PPV VP2 gene laid the basis for further studies on patho- oenesis, early clinical diaonosis of this virus and quantitative analysis of PPV infection.
基金Supported by National Key Technology R&D Program (2006BAD06A12)Gansu Agricultural Biotechnology Research and Application Development Project (GNSW-2005-17)~~
文摘Three pairs of primer were designed for amplification of porcine pseudorabies virus （PRV） gB, gE, and TK gene by multiplex PCR （multi-PCR） in order to differentiate vaccine strains from field isolates. Three specific bands were obtained respectively at the expected size, 427 bp （gB gene）, 298 bp （gEgene）, and 208 bp （TKgene）, Then four different gene-deleted vaccines of PRV were detected by multi-PCR. One ex- pected specific band was observed in one of samples, while two bands in the others. As shown by the detection results, the multi-PCR has high sensitivity and specificity and should be applied in pathogen diagnosis and epidemiological investigation in the future.
