克隆化探针中载体与临床样品同源性的研究  

Study on Vector Homology in Clinical Specimens with Cloned Probes

作　　者：伍欣星[1] 杨平[1] 赵文先[1] 刘薇茜[1] 谭云[1] 

机构地区：[1]湖北医学院病毒研究所生化室

出　　处：《病毒学杂志》1989年第3期239-244,共6页

基　　金：国家自然科学基金资助项目

摘　　要：当前,克隆化探针(由病毒核酸片段和细菌质粒一载体组成)在临床样品的实验诊断和研究中得到了应用。它具有简便、快速、灵敏等优点,并有助于检测那些难于或不能培养的病毒。但是克隆化探针中载体与临床样品的同源性问题应引起重视。我们用常见的克隆载体pBR322作探针,用核酸点杂交和Southern吸印方法,检测了来自173人的213例样品。结果表明,在常用的临床样品:人宫颈脱落细胞、宫颈活检组织、血液中都含有pBR322 DNA的同源序列,这就意味着如果用克隆化探针检测这些临床样品时,可能出现样品与载体发生非特异性反应,而不是与病毒基因片段的特异性反应。实验还表明,经一次电泳纯化的病毒基因片段与pBR322探针杂交,阳性信号明显减弱,但没有完全消失,说明仍有少量pBR322污染。因此,在运用克隆化探针检测临床各类样品时,各系统要按常规设载体探针对照,或者用多次纯化的分离病毒基因片段作探针,以排除载体与临床样品的同源性问题。Nucleic acid hybridization techniques using cloned probes (recombinantplasmids composed of viral fragment and bacterial plasmid vector, such as pBR322)are applied in assay of clinical specimen in research and diagnostic laborato-ries. They have simple, quick and sensitive adventages, and help to detectthose virus gene that are difficult or impossible to culture. However, it should be noted that there may be a homology problem between cloued probe and cli-nical specimens. We detected 213 specimens from 173 people using probe of pBR-322 by Dot and Southern blot hybridization. It is shown that there was materialhomologous to pBR322 present in many clinical samples: cervical scrapes andDNA, cervical biopsies and blood. It means that a hybridization may presentbetween semples and vector, other than virous gene, if using cloned probe to detectthese samples. It is also shown that the relative intensity of hybridization was muchreduced but not eliminated viral fragment isolated by a single gel electropho-resis to pBR322 probe. This implies that non-special reaction can appear onheavy bacterial contaminant. Therefore, it is essential that all detection sys-tems use a control probe of vector alone or the repetitive gel isolated fragmentas a probe in hybridization studies to demonstrate the absence of material withvector homology in the sample tested.This is the first report in China.

关 键 词：载体同源性 分子杂交 克隆化探针 

分 类 号：R446.62[医药卫生—诊断学]