马立克病毒基因缺失疫苗与商品化疫苗的分子鉴别方法

发布时间：2018-01-22 02:38

本文关键词： 马立克病毒(MDV) SC- 商品化疫苗 PCR扩增斑点杂交　出处：《病毒学报》2017年04期 　论文类型：期刊论文

【摘要】：为了对马立克病毒(MDV)meq基因缺失疫苗株SC9-1与商品化MD疫苗CVI988/Rispens株、814株和HVT FC-126株进行分子鉴别,本研究根据疫苗株SC9-1与其它MD疫苗株的基因组差异,分别针对I型MDVmeq基因以及SC9-1基因组中的禽网状内皮组织增殖症病毒-长末端重复序列(REV-LTR)插入片段,设计三对PCR扩增引物,同时合成针对meq基因和REV-LTR片段的地高辛标记探针,通过PCR扩增和斑点杂交方法区分SC9-1株与其他疫苗株。结果表明,使用针对meq基因的引物F1/R1进行PCR扩增,SC9-1株、CVI988/Rispens株、814株分别扩增出184bp、1 297bp、1 297bp目的片段,HVT FC-126株没有条带;使用针对meq基因的引物F2/R2进行PCR扩增,CVI988/Rispens株、814株均扩增出746bp目的片段,SC9-1株、HVT FC-126株均没有条带;使用针对REV-LTR片段设计的引物F3/R3进行PCR扩增,SC9-1株扩增出512bp目的片段,CVI988/Rispens株、814株、HVT FC-126株均没有条带。同时,使用针对meq基因的探针进行斑点杂交,CVI988/Rispens株、814株均显示阳性结果,SC9-1株、HVT FC-126株均为阴性;使用针对REV-LTR的探针进行斑点杂交,SC9-1株显示阳性结果,CVI988/Rispens株、814株、HVT FC-126株均为阴性。因此,利用本研究设计的特异性PCR引物以及探针,通过PCR扩增和斑点杂交方法可有效区分SC9-1与其他商品化MD疫苗。
[Abstract]:In order to detect the MDV meq gene deletion vaccine strain (SC9-1) and the commercial MD vaccine strain (CVI988/Rispens) of Marek virus. 814 strains and HVT FC-126 strains were identified by molecular analysis. In this study, the genomic differences between the vaccine strain SC9-1 and other MD vaccine strains were studied. The insertion fragments of type I MDVmeq gene and the long terminal repeat sequence of reticuloendothelial cell proliferation virus (reticuloendotheliavirus) in the SC9-1 genome were inserted respectively. Three pairs of PCR amplification primers were designed and digoxigenin labeled probes were synthesized for both meq gene and REV-LTR fragment. SC9-1 strain was distinguished from other vaccine strains by PCR amplification and dot blot hybridization. The results showed that the primers F1 / R1 for meq gene were used for PCR amplification of SC9-1 strain. 1844 CVI988/Rispens strains were amplified from 1849 BP 1 297 BP FC-126 strain, and there were no bands in the FC-126 strain. Using the primers F2 / R2 of meq gene to amplify the 746bp target fragment of SC9-1 strain by PCR amplification of CVI988% Rispens strain (814 strains). There were no bands in HVT FC-126 strains. Using the primers F3 / R3 designed for REV-LTR fragment, 512bp target fragment of CVI988 / Rispens strain was amplified by PCR amplification of SC9-1 strain. There were no bands in 814 meq FC-126 strains. Meanwhile, meq gene probes were used for dot blot hybridization of CVI988 / Rispens strain. All the 814 strains showed positive results. All the SC9-1 FC-126 strains were negative. Dot blot hybridization of SC9-1 strain against REV-LTR showed positive results of 814 CVI988 / Rispens strains. HVT FC-126 strains were all negative. Therefore, the specific PCR primers and probes designed in this study were used. PCR amplification and dot blot hybridization can effectively distinguish SC9-1 from other commercial MD vaccines.
【作者单位】：山东农业大学动物科技学院;
【基金】：国家自然科学基金河南省联合基金(项目号:U1604232),题目:鸡马立克病病毒(MDV)遗传、变异与致病的分子机制~~
【分类号】：S852.4
【正文快照】： 马立克病(Marek's disease,MD)是由马立克病毒(Marek's disease virus,MDV)引起的一种鸡的肿瘤性疾病,病毒可经空气、灰尘传播,具有较强传染力[1],给鸡群带来严重危害。MD疫苗由非致病性的III型MDV HVT FC-126株逐渐发展到传代致弱的I型MDV CVI988/Rispens株[2,3],虽然国内研

【相似文献】