为研究从猪血清中分离的猪圆环病毒2型（Porcine circovirus type 2,PCV-2）与从发病猪组织中分离的PCV-2之间的差异、了解贵州地区PCV-2遗传变异情况、丰富贵州PCV-2全基因组序列库,试验采用来自贵州省毕节市金沙县、遵义市绥阳县、毕...为研究从猪血清中分离的猪圆环病毒2型（Porcine circovirus type 2,PCV-2）与从发病猪组织中分离的PCV-2之间的差异、了解贵州地区PCV-2遗传变异情况、丰富贵州PCV-2全基因组序列库,试验采用来自贵州省毕节市金沙县、遵义市绥阳县、毕节市黔西县和贵阳市修文县的PCV-2抗体阳性猪血清,通过PCR扩增及通过T克隆获得了4株PCV-2全基因,分别命名为GZ-JS2015、GZSY2016、GZ-QX2014和GZ-XW2014,并进行测序;将测序结果与笔者收集的38株PCV-2的核苷酸序列进行比对分析。结果表明：在病毒血症期间猪血清中分离的PCV-2与从发病猪组织中分离的PCV-2并无太大差异;笔者收集的血清中贵州省为10株,其中8株为PCV-2b型、2株为PCV-2a型,说明在贵州地区流行的主要是PCV-2b型;来自贵州省同一地区的2株PCV-2处于不同的进化树分支上,与省外的PCV-2分离株亲缘关系较近,提示在贵州省流行的部分PCV-2可能由引种时流入。展开更多
[Objective] In this study, the quantitive detection of PCV2 （porcine circovirus type 2） in vitro was achieved. We aimed to establish two kinds of TaqMan real-time PCR methods based on PCV20RF1 and ORF2 respectively ...[Objective] In this study, the quantitive detection of PCV2 （porcine circovirus type 2） in vitro was achieved. We aimed to establish two kinds of TaqMan real-time PCR methods based on PCV20RF1 and ORF2 respectively and compare them. [Method] According to the relatively＇conserved sequences of PCV20RF1 and ORF2 registered in GenBank, two pairs of specific primers and TaqMan probes were designed and synthesized. Then the recombinant plasmids containing the whole sequences of PCV20RF1 and ORF2 were constructed to draw the standard curves through optimizing the reaction system and conditions. And thus two kinds of TaqMan real-time PCR detection methods based on the whole sequences of ORF1 and ORF2 respectively were constructed for PCV2. [Result] For the two established standard curves, the Ct values showed a good linear relationship with the loga- rithms of copy numbers of templates （F2〉0.99）. The amplification efficiency ranged from 90% to 110%. The amplifications all had a good repeatability with variation coefficients within groups all less than 5%. Moreover, the amplifications all had a good specificity. When the sequences of porcine parvovirus （PPV）, porcine circovirus type 1 （PCV1）, swine pseudorabies virus （PRV）, porcine reproductive and respiratory syndrome virus （PRRSV） were used as templates, the target sequence was not amplified. The amplifications also had a high sensitivity. The ORF1 detection method could reach 1.0x10T copies/;ul, and the ORF2 detection method could reach 1.0×10^2 copies/μl. The two established real-time PCR detection methods were used to detect the 80 clinical samples respectively. The results showed the magnitudes of 72 amplified samples were basically consistent between the 2 detection methods, while the magnitudes of the other 8 amplified samples were inconsistent. Then the 8 samples were detected with SYBR Green I real-time PCR method established based on the sequence of PCV2-1ike factor P1 by Wen et aL The PCV2-1ike factor P1 was amplified in all the 8 samples, indicating the 8 samples were all infected with PCV2-1ike factor P1. [Conclusion] The ORFl-based detection method has a higher accuracy, and it can be used for the rapid detection of PCV2.展开更多
文摘为研究从猪血清中分离的猪圆环病毒2型（Porcine circovirus type 2,PCV-2）与从发病猪组织中分离的PCV-2之间的差异、了解贵州地区PCV-2遗传变异情况、丰富贵州PCV-2全基因组序列库,试验采用来自贵州省毕节市金沙县、遵义市绥阳县、毕节市黔西县和贵阳市修文县的PCV-2抗体阳性猪血清,通过PCR扩增及通过T克隆获得了4株PCV-2全基因,分别命名为GZ-JS2015、GZSY2016、GZ-QX2014和GZ-XW2014,并进行测序;将测序结果与笔者收集的38株PCV-2的核苷酸序列进行比对分析。结果表明：在病毒血症期间猪血清中分离的PCV-2与从发病猪组织中分离的PCV-2并无太大差异;笔者收集的血清中贵州省为10株,其中8株为PCV-2b型、2株为PCV-2a型,说明在贵州地区流行的主要是PCV-2b型;来自贵州省同一地区的2株PCV-2处于不同的进化树分支上,与省外的PCV-2分离株亲缘关系较近,提示在贵州省流行的部分PCV-2可能由引种时流入。
基金Supported by National Natural Science Foundation of China(31302071)Special Fund for Agro-scientific Research in the Public Interest(201303046)+1 种基金Jiangsu Agricultural Science and Technology Innovation Fund(CX(14)2045)"333 High-level Personnel Training Project"of Jiangsu Province(BRA2012194)~~
文摘[Objective] In this study, the quantitive detection of PCV2 （porcine circovirus type 2） in vitro was achieved. We aimed to establish two kinds of TaqMan real-time PCR methods based on PCV20RF1 and ORF2 respectively and compare them. [Method] According to the relatively＇conserved sequences of PCV20RF1 and ORF2 registered in GenBank, two pairs of specific primers and TaqMan probes were designed and synthesized. Then the recombinant plasmids containing the whole sequences of PCV20RF1 and ORF2 were constructed to draw the standard curves through optimizing the reaction system and conditions. And thus two kinds of TaqMan real-time PCR detection methods based on the whole sequences of ORF1 and ORF2 respectively were constructed for PCV2. [Result] For the two established standard curves, the Ct values showed a good linear relationship with the loga- rithms of copy numbers of templates （F2〉0.99）. The amplification efficiency ranged from 90% to 110%. The amplifications all had a good repeatability with variation coefficients within groups all less than 5%. Moreover, the amplifications all had a good specificity. When the sequences of porcine parvovirus （PPV）, porcine circovirus type 1 （PCV1）, swine pseudorabies virus （PRV）, porcine reproductive and respiratory syndrome virus （PRRSV） were used as templates, the target sequence was not amplified. The amplifications also had a high sensitivity. The ORF1 detection method could reach 1.0x10T copies/;ul, and the ORF2 detection method could reach 1.0×10^2 copies/μl. The two established real-time PCR detection methods were used to detect the 80 clinical samples respectively. The results showed the magnitudes of 72 amplified samples were basically consistent between the 2 detection methods, while the magnitudes of the other 8 amplified samples were inconsistent. Then the 8 samples were detected with SYBR Green I real-time PCR method established based on the sequence of PCV2-1ike factor P1 by Wen et aL The PCV2-1ike factor P1 was amplified in all the 8 samples, indicating the 8 samples were all infected with PCV2-1ike factor P1. [Conclusion] The ORFl-based detection method has a higher accuracy, and it can be used for the rapid detection of PCV2.
