【目的】确定广东某规模化猪场发病猪是否为猪圆环病毒2型(Porcine circovirus type 2,PCV2)感染,并评估病毒的遗传特性及其在猪群中的流行情况,为疫病的防控提供科学数据支持。【方法】采用实时荧光定量PCR和ELISA方法对猪场病猪的肾...【目的】确定广东某规模化猪场发病猪是否为猪圆环病毒2型(Porcine circovirus type 2,PCV2)感染,并评估病毒的遗传特性及其在猪群中的流行情况,为疫病的防控提供科学数据支持。【方法】采用实时荧光定量PCR和ELISA方法对猪场病猪的肾脏、淋巴结、脾脏等内脏组织及血清样本进行检测分析,并通过PCR扩增PCV2阳性样本的ORF2基因。利用DNAStar软件包中的MegAlign模块对所得ORF2基因序列与GenBank数据库中23株PCV2参考毒株进行相似性比对。运用Mega 7.0软件构建基于ORF2基因序列的遗传进化树,以明确鉴定毒株与参考毒株之间的遗传关系。【结果】流行病学调查、临床症状及病理解剖结果显示,该猪场病猪符合PCV2感染的特征。实时荧光定量PCR检测发现3份样本的Ct值分别为14.42、15.13和16.08,表明病毒载量较高,且PCV2检测结果为阳性。ELISA检测结果显示,不同日龄段猪群中Cap蛋白抗体阳性率介于60%~100%之间,而Rep蛋白抗体阳性率则在0~100%之间,特别是150日龄猪群的2种蛋白抗体阳性率均达到100%,表明PCV2感染强度较高。ORF2基因测序获得了3条序列,分别命名为LZC-2305-1、LZC-2305-2和LZC-2305-3。相似性分析结果显示,这3条序列间具有99.9%~100%的相似性,且与GenBank中的23株PCV2参考毒株序列的相似性为82.3%~99.7%,其中与CZ246序列相似性最高,为99.6%~99.7%。遗传进化树分析进一步证实,本研究获得的3条序列与CZ246序列具有最近的亲缘关系,归属于PCV2的2d分支。【结论】本研究确诊该猪场猪群发生了PCV2d分支的感染。本研究结果不仅为该猪场的PCV2防控提供了科学依据,也为其他规模化猪场的疫病监测与防控提供了参考。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【目的】确定广东某规模化猪场发病猪是否为猪圆环病毒2型(Porcine circovirus type 2,PCV2)感染,并评估病毒的遗传特性及其在猪群中的流行情况,为疫病的防控提供科学数据支持。【方法】采用实时荧光定量PCR和ELISA方法对猪场病猪的肾脏、淋巴结、脾脏等内脏组织及血清样本进行检测分析,并通过PCR扩增PCV2阳性样本的ORF2基因。利用DNAStar软件包中的MegAlign模块对所得ORF2基因序列与GenBank数据库中23株PCV2参考毒株进行相似性比对。运用Mega 7.0软件构建基于ORF2基因序列的遗传进化树,以明确鉴定毒株与参考毒株之间的遗传关系。【结果】流行病学调查、临床症状及病理解剖结果显示,该猪场病猪符合PCV2感染的特征。实时荧光定量PCR检测发现3份样本的Ct值分别为14.42、15.13和16.08,表明病毒载量较高,且PCV2检测结果为阳性。ELISA检测结果显示,不同日龄段猪群中Cap蛋白抗体阳性率介于60%~100%之间,而Rep蛋白抗体阳性率则在0~100%之间,特别是150日龄猪群的2种蛋白抗体阳性率均达到100%,表明PCV2感染强度较高。ORF2基因测序获得了3条序列,分别命名为LZC-2305-1、LZC-2305-2和LZC-2305-3。相似性分析结果显示,这3条序列间具有99.9%~100%的相似性,且与GenBank中的23株PCV2参考毒株序列的相似性为82.3%~99.7%,其中与CZ246序列相似性最高,为99.6%~99.7%。遗传进化树分析进一步证实,本研究获得的3条序列与CZ246序列具有最近的亲缘关系,归属于PCV2的2d分支。【结论】本研究确诊该猪场猪群发生了PCV2d分支的感染。本研究结果不仅为该猪场的PCV2防控提供了科学依据,也为其他规模化猪场的疫病监测与防控提供了参考。
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.
