| 鉴别犬瘟热病毒疫苗株和野毒株RT—PCR—RFLP方法的建立及应用 |
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| 引用本文： | 张洪亮,张传美,王聪,黄娟,任颖超,单虎.鉴别犬瘟热病毒疫苗株和野毒株RT—PCR—RFLP方法的建立及应用[J].兽医大学学报,2013(11):1647-1656. |
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| 作者姓名： | 张洪亮 张传美 王聪 黄娟 任颖超 单虎 |
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| 作者单位： | 青岛农业大学山东省预防兽医学重点实验室,山东青岛266109 |
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| 基金项目： | 山东省自然科学基金（ZR2010CL023）;科技基础性工作专项（2012FY111000）;863计划（201IAAi006）0703 |
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| 摘 要： | 根据Onderstepoort株H基因序列，设计1对引物建立RT-PCR-RFLP检测方法，对不同宿主来源的疑似犬瘟热临床样品进行检测，并对检出的犬瘟热病毒野毒山东株PCR产物进行克隆和序列分析，验证RT—PCR—RFLP检测方法。结果RT—PCR扩增片段为1921bp，产物经RFLP分析，野毒株的PCR产物能被NdeI酶切为1282、345和294bp3个片段，弱毒疫苗株则不能被切开；病毒RNA的最小检出量为2．15ng。临床检测共检出19份样品为犬瘟热阳性，其中15份为野毒株感染，其H基因编码区全长为1824bp，均在1279和1543处有NdeI酶切位点，推导氨基酸与野毒株的同源性在92．9％～96．9％，与疫苗株的同源性在89．0％～90．8％，进化树分析显示这些毒株在基因型上属于Asia-1型，为野毒株谱系。该方法的建立为临床上犬瘟热病毒的鉴别检测和诊断提供了依据。
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| 关 键 词： | 犬瘟热病毒 鉴别方法 RT—PCR-RFLP 血凝素蛋白（H） |
Development of RT-PCR-RFLP for detection and differentiation of wild-type and vaccine viruses of canine distemper virus |
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| Authors: | ZHANG Hong-liang ZHANG Chuan-mei WANG Hu Shan |
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| Institution: | ZHANG Hong-liang, ZHANG Chuan-mei , WANG Hu (Shandong Key Laboratory of Preventive niversity , Qingdao, Shandong 266109, China) Cong, HUANG Juan , REN Yin-chao, SHAN Veterinary Medicine, Qingdao Agricultural U- |
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| Abstract: | In order to develop a differentiation method for detection of wild-type and vaccine viru- ses of canine distemper virus（CDV）,a pair of primers was designed according to the hemaggluti- nin（H） gene sequence of Onderstepoort strain,and RT-PCR-RFLP was developed and used to de- tect suspected clinical samples collected from different host-species in Shandong province during 2010--2011. PCR products of wild-type CDV were subjected to gene sequencing. The results showed that a fragment of 1 921 bp was amplified from wild-type CDV which could be digested with Nde II into three fragments,while the vaccine viruses could not be digested with Nde I ,the lowest detected concentration of RNA was 2.15 ng. Nineteen positive samples of CD were detec- ted by RT-PCR,among of which, 15 wild-type strains identified by RT-PCR-RFLP contained two Nde I recognition sequence at site 1 279 and 1 543 of 1 824 bp-length H gene coding region,the deduced amino acid of 15 field strains showed 92.9%-96.9% homology with those of refered wild- type strains,and only 89.0%-90.8% identity with those of refered vaccine virus,the genotype of 15 field strains revealed by phylogenetic analysis was Asia-I type which belongs to wild-strain pedigree. The developed RT-PCR-RFLP could be used to detect and differentiate wild-type CDV from vaccine strains. |
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| Keywords: | Canine distemper virus Differential assay RT-PCR-RFLP Hemagglutinin（H） |
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