为建立H3亚型犬流感病毒(CIV)荧光定量PCR检测方法,本研究根据H3亚型CIV HA基因的保守区序列,设计合成一对特异性引物和Taq Man MGB探针,经条件优化后初步建立了H3亚型CIV荧光定量RT-PCR检测方法。结果显示,构建的重组质粒标准品在109拷...为建立H3亚型犬流感病毒(CIV)荧光定量PCR检测方法,本研究根据H3亚型CIV HA基因的保守区序列,设计合成一对特异性引物和Taq Man MGB探针,经条件优化后初步建立了H3亚型CIV荧光定量RT-PCR检测方法。结果显示,构建的重组质粒标准品在109拷贝/μL~10^(2)拷贝/μL浓度范围内有良好的线性关系,标准曲线的相关系数为0.999。该方法仅能特异性扩增H3N2亚型CIV核酸,与H3N2亚型禽流感病毒、人流感病毒和猪流感病毒及H9N2亚型CIV、H1N1亚型猪流感病毒、犬腺病毒、犬瘟热病毒、犬细小病毒、犬冠状病毒等病毒核酸均无交叉反应。该方法最低可检测到的质粒标准品浓度为19拷贝/μL,敏感性高于普通PCR方法。组内与组间重复性试验的变异系数均小于1%。利用该方法对83份临床样品的核酸进行检测,检测结果与病毒分离结果总符合率为97.59%。本研究建立的荧光定量PCR检测方法特异性强、敏感性高、重复性好、操作简便,为临床H3亚型CIV的快速检测提供可靠方法。展开更多
In the past decade,there has been extensive global surveillance for highly pathogenic avian influenza(HPAI)infection in both animals and humans,however,few studies on epidemiology of avian influenza in Democratic Peo...In the past decade,there has been extensive global surveillance for highly pathogenic avian influenza(HPAI)infection in both animals and humans,however,few studies on epidemiology of avian influenza in Democratic People’s Republic of Korea(DPRK)were published.During the period 2013–2014,HPAI H5N1 viruses were detected with outbreaks in domestic poultry in DPRK.Phylogenetic analysis revealed that the hemagglutinin gene of all samples belonged to clade 2.3.2.1c with high homology.The HPAI H5N1 virus found in ducks at the Tudan Duck Farm in 2013 was might introduced by migratory birds and then led to the outbreaks on neighboring chicken farms in 2014.These data provide direct evidence for the transmission of avian influenza viruses from wild birds to waterfowl to terrestrial birds.Therefore,the monitoring and control of influenza virus in ducks must be given top priority,which are essential components to prevent and control HPAI.展开更多
The HA1 gene of H3N2 subtype swine influenza virus(SIV)was cloned into the expression plasmid pET-30a,the recombinant plasmid was named pET-HAl.This was transformed into E.coli BL21(DE3),and expressed by induction wit...The HA1 gene of H3N2 subtype swine influenza virus(SIV)was cloned into the expression plasmid pET-30a,the recombinant plasmid was named pET-HAl.This was transformed into E.coli BL21(DE3),and expressed by induction with IPTG.The expressed HA protein was identified by SDS-PAGE and Western blotting which showed the protein to be 42kDa and was immunoreactive.The purified HA protein was used to establish the indirect ELIS A for detection of the antibodies,specifically against the H3 subtype of SIV.The assay has excellent specificity,sensitivity and reproducibility.When 96 serum samples,randomly collected from the field,were evaluated in parallel by this new ELISA using recombinant HA1 and a routine HI test,the coincidental rate between the two tests was 86.5%.These results show that the recombinant HAl-based ELISA is specific,sensitive and easy to perform for the serological diagnosis of SIV infection.展开更多
To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defectiv...To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defective recombinant adenovirus expressing the HA gene(rAd-H5HA-EGFP) was generated by co-transfecting the recombinant shuttle plasmid pDC315-H5HA-EGFP and the genomic plasmid pBHGlox△E1,E3Cre in HEK293 cells.The recombinant adenovirus was confirmed by PCR,RT-PCR and Western blot assay.These results demonstrated that HA protein was properly expressed by the rAd-H5HA-EGFP in HEK293 cells and had natural biological activities.The TCID<sub>50</sub> of the rAd-H5HA- EGFP was assessed to be 2.26×10<sup>10</sup>/mL after propagation and purification.Immunization of BALB/ c mice indicated that rAd-H5HA-EGFP induced HI antibodies and protected mice from replication of the challenge virus in their lungs.展开更多
文摘为建立H3亚型犬流感病毒(CIV)荧光定量PCR检测方法,本研究根据H3亚型CIV HA基因的保守区序列,设计合成一对特异性引物和Taq Man MGB探针,经条件优化后初步建立了H3亚型CIV荧光定量RT-PCR检测方法。结果显示,构建的重组质粒标准品在109拷贝/μL~10^(2)拷贝/μL浓度范围内有良好的线性关系,标准曲线的相关系数为0.999。该方法仅能特异性扩增H3N2亚型CIV核酸,与H3N2亚型禽流感病毒、人流感病毒和猪流感病毒及H9N2亚型CIV、H1N1亚型猪流感病毒、犬腺病毒、犬瘟热病毒、犬细小病毒、犬冠状病毒等病毒核酸均无交叉反应。该方法最低可检测到的质粒标准品浓度为19拷贝/μL,敏感性高于普通PCR方法。组内与组间重复性试验的变异系数均小于1%。利用该方法对83份临床样品的核酸进行检测,检测结果与病毒分离结果总符合率为97.59%。本研究建立的荧光定量PCR检测方法特异性强、敏感性高、重复性好、操作简便,为临床H3亚型CIV的快速检测提供可靠方法。
基金supported by the China Agriculture Research System of MOF and MARA(CARS-41)。
文摘In the past decade,there has been extensive global surveillance for highly pathogenic avian influenza(HPAI)infection in both animals and humans,however,few studies on epidemiology of avian influenza in Democratic People’s Republic of Korea(DPRK)were published.During the period 2013–2014,HPAI H5N1 viruses were detected with outbreaks in domestic poultry in DPRK.Phylogenetic analysis revealed that the hemagglutinin gene of all samples belonged to clade 2.3.2.1c with high homology.The HPAI H5N1 virus found in ducks at the Tudan Duck Farm in 2013 was might introduced by migratory birds and then led to the outbreaks on neighboring chicken farms in 2014.These data provide direct evidence for the transmission of avian influenza viruses from wild birds to waterfowl to terrestrial birds.Therefore,the monitoring and control of influenza virus in ducks must be given top priority,which are essential components to prevent and control HPAI.
基金supported by the Chinese National S&T Plan(2004BA519A55)
文摘The HA1 gene of H3N2 subtype swine influenza virus(SIV)was cloned into the expression plasmid pET-30a,the recombinant plasmid was named pET-HAl.This was transformed into E.coli BL21(DE3),and expressed by induction with IPTG.The expressed HA protein was identified by SDS-PAGE and Western blotting which showed the protein to be 42kDa and was immunoreactive.The purified HA protein was used to establish the indirect ELIS A for detection of the antibodies,specifically against the H3 subtype of SIV.The assay has excellent specificity,sensitivity and reproducibility.When 96 serum samples,randomly collected from the field,were evaluated in parallel by this new ELISA using recombinant HA1 and a routine HI test,the coincidental rate between the two tests was 86.5%.These results show that the recombinant HAl-based ELISA is specific,sensitive and easy to perform for the serological diagnosis of SIV infection.
基金supported by the Chinese National S&T Plan(2004BA519A55)Scientific Research Program of State Key Laboratory of Veterinary Biotechnology(NKLVBP200818)
文摘To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defective recombinant adenovirus expressing the HA gene(rAd-H5HA-EGFP) was generated by co-transfecting the recombinant shuttle plasmid pDC315-H5HA-EGFP and the genomic plasmid pBHGlox△E1,E3Cre in HEK293 cells.The recombinant adenovirus was confirmed by PCR,RT-PCR and Western blot assay.These results demonstrated that HA protein was properly expressed by the rAd-H5HA-EGFP in HEK293 cells and had natural biological activities.The TCID<sub>50</sub> of the rAd-H5HA- EGFP was assessed to be 2.26×10<sup>10</sup>/mL after propagation and purification.Immunization of BALB/ c mice indicated that rAd-H5HA-EGFP induced HI antibodies and protected mice from replication of the challenge virus in their lungs.
