The 5′ nonencoding region, p23 and p14 encoding region and E1 gene of hog cholera virus (HCV) strain C were amplified from total RNA extracted from HCV strain C infected rabbit spleen by reverse transcription and nes...The 5′ nonencoding region, p23 and p14 encoding region and E1 gene of hog cholera virus (HCV) strain C were amplified from total RNA extracted from HCV strain C infected rabbit spleen by reverse transcription and nested or half\_nested PCR. The PCR products were cloned into pGEM\|T vector. Nucleotide sequencing was performed using an ABI PRISM sequencing device; based on the incorporation of fluoresect labelled dideoxynuclotide teminators. The obtained sequences on 5′ noncoding region and part of p23 and p14 encoding region were compared with HCV strains Shimen and C sequenced in Moormann’s lab. The result showed that the homology between HCV strains C sequenced in this report and in Moormann’s lab was 99.19%, and the homology between HCV strains Shimen, the standard virulent HCV strain in China, and C sequenced in this report was 94.69%. It was also discovered that the base C at 244 of the genome of HCV strains Shimen and C sequenced in this report was absent at the genome of C strain sequenced in Moormann’s lab et al.展开更多
根据GenBank中登录的猪瘟病毒(CSFV)野毒株、猪瘟兔化弱毒疫苗(HCLV)和牛病毒性腹泻病毒(BVDV)的NS5B基因序列,利用在线软件Pri mer Explorer V4Software,设计了一套环介导逆转录等温核酸扩增(RT-LAMP)引物,建立了特异性检测HCLV的RT-L...根据GenBank中登录的猪瘟病毒(CSFV)野毒株、猪瘟兔化弱毒疫苗(HCLV)和牛病毒性腹泻病毒(BVDV)的NS5B基因序列,利用在线软件Pri mer Explorer V4Software,设计了一套环介导逆转录等温核酸扩增(RT-LAMP)引物,建立了特异性检测HCLV的RT-LAMP方法。在BstDNA聚合酶作用下,65℃恒温反应1h即可完成扩增过程,扩增产物可通过20g/L琼脂糖凝胶电泳或加入SYBR GreenⅠ染料进行分析。经检测,该方法对于CSFV、BVDV以及其他猪源病毒无特异性扩增,具有良好的HCLV特异性;其灵敏度与本实验室建立的TaqMan实时荧光定量RT-PCR检测结果一致,均达到5个拷贝。分别用这两种方法对58份市售猪瘟弱毒疫苗进行检测,结果符合率达100%。表明,建立的HCLV RT-LAMP技术可以应用于基层实验室或养殖场,便于养殖户对疫苗中的弱毒含量进行监测。展开更多
文摘The 5′ nonencoding region, p23 and p14 encoding region and E1 gene of hog cholera virus (HCV) strain C were amplified from total RNA extracted from HCV strain C infected rabbit spleen by reverse transcription and nested or half\_nested PCR. The PCR products were cloned into pGEM\|T vector. Nucleotide sequencing was performed using an ABI PRISM sequencing device; based on the incorporation of fluoresect labelled dideoxynuclotide teminators. The obtained sequences on 5′ noncoding region and part of p23 and p14 encoding region were compared with HCV strains Shimen and C sequenced in Moormann’s lab. The result showed that the homology between HCV strains C sequenced in this report and in Moormann’s lab was 99.19%, and the homology between HCV strains Shimen, the standard virulent HCV strain in China, and C sequenced in this report was 94.69%. It was also discovered that the base C at 244 of the genome of HCV strains Shimen and C sequenced in this report was absent at the genome of C strain sequenced in Moormann’s lab et al.
文摘根据GenBank中登录的猪瘟病毒(CSFV)野毒株、猪瘟兔化弱毒疫苗(HCLV)和牛病毒性腹泻病毒(BVDV)的NS5B基因序列,利用在线软件Pri mer Explorer V4Software,设计了一套环介导逆转录等温核酸扩增(RT-LAMP)引物,建立了特异性检测HCLV的RT-LAMP方法。在BstDNA聚合酶作用下,65℃恒温反应1h即可完成扩增过程,扩增产物可通过20g/L琼脂糖凝胶电泳或加入SYBR GreenⅠ染料进行分析。经检测,该方法对于CSFV、BVDV以及其他猪源病毒无特异性扩增,具有良好的HCLV特异性;其灵敏度与本实验室建立的TaqMan实时荧光定量RT-PCR检测结果一致,均达到5个拷贝。分别用这两种方法对58份市售猪瘟弱毒疫苗进行检测,结果符合率达100%。表明,建立的HCLV RT-LAMP技术可以应用于基层实验室或养殖场,便于养殖户对疫苗中的弱毒含量进行监测。
