摘要
为了建立烟草花叶病毒辽宁分离物(TMV-LN)的高特异性、高灵敏度的分子杂交检测体系,从粗提纯的TMV-LN粒子中提取RNA,设计特异性引物通过RT-PCR扩增TMV-LN的CP和3'端非翻译区域,将片段连至p UC119载体获得重组质粒p UCTMV-PP,体外转录获得地高辛(DIG)标记的TMV-LN正义链RNA杂交检测探针,同时构建DNA检测探针作为对照。采用点印迹(Dot-blot)杂交和Northern杂交对比RNA探针和DNA探针对TMV-LN的检测特异性和灵敏性。检测结果表明,RNA探针和DNA探针在点印记杂交和Northern杂交中均表现出良好的检测特异性,RNA探针在检测灵敏度方面要略好于DNA探针,且点印迹杂交体系在病毒定性方面较为快捷,Northern杂交体系在病毒基因组RNA的定量方面具有明显优势。
In order to establish a more sensitive and specific molecular hybridization detection system of Tobacco mosaic virus Liaoningisolate（TMV-LN）,total viral RNA was extracted from purified TMV-LN viral particles and amplified by reverse-transcript PCR. The PCRproducts were ligated into p UC119 vector to construct p UCTMV-PP that express RNA detection probe using Digoxigenin（DIG）NorthernStarter Kit. DNA probe was also constructed using DIG DNA hybridization kit. Dot-blot hybridization and Northern blot hybridization analysiswere performed to study the specificity and sensitivity of the RNA probe and DNA probe constructed above. Both of the probes showed highspecificity and sensitivity in TMV-LN detection through Dot-blot hybridization and Northern blot hybridization detection system. Comparison oftwo hybridization systems,Dot-blot hybridization system is suitable for virus qualitative detection,whereas,Northern blot hybridization hasits advantage in relative quantification of viral genome RNA.
作者
李艳丽
安梦楠
王冠中
吴元华
LI Yan-li AN Meng-nan WANG Guan-zhong WU Yuan-hua(College of Plant Protection, Shenyang Agricultural University, Shenyang 110161)
出处
《生物技术通报》
CAS
CSCD
北大核心
2017年第4期137-142,共6页
Biotechnology Bulletin
基金
国家自然科学基金项目(31401710)
国家烟草专卖局科技项目[110201601024(LS-04)]
