反义RNA重组逆转录病毒逆转录套式PCR检测方法的建立  

Development of RT-nested PCR for detection of antisense RNA recombinant retrovirus

作　　者：曹素芳[1] 孟庆文[2] 于康震[2] 单文鲁[3] 

机构地区：[1]郑州牧业工程高等专科学校,河南郑州450011 [2]中国农业科学院哈尔滨兽医研究所,黑龙江哈尔滨150001 [3]新疆农业大学动物医学院,新疆乌鲁木齐830052

出　　处：《中国兽医科技》2005年第8期611-614,共4页Chinese Journal of Veterinary Science and Technology

基　　金：国家重点基础研究发展规划(973)项目(G199901195)

摘　　要：为探求一种快速检测反义RNA重组逆转录病毒效价的新方法,用脂质体介导法将反义RNA重组逆转录病毒载体pLXSA、pLXSB分别转染包装细胞系PA317,经G418筛选,获得数个稳定的产毒细胞克隆,择优挑选7个细胞克隆扩增培养,收集细胞上清,用异硫氰酸胍-酚一步法提取病毒RNA,进行逆转录套式PCR,检测细胞上清中的反义RNA重组逆转录病毒效价,同时用传统方法(小鼠成纤维放大法)进行测定。2种方法检测结果的比较表明,逆转录套式PCR法的特异性和敏感性均高于小鼠成纤维放大法,是一种敏感性高、特异性强、操作简便、快速的检测逆转录病毒效价的新方法。In order to develop a rapid and sensitive method for detection of antisense RNA recombinant retrovirus titers, antisense RNA recombinant retrovirus vectors pLXSA and pLXSB were packed by packaging cell lines PA317 and screened by G418. Several stable cell clones producing recombinant retrovirus were obtained. Seven selected clones were expanded. The RNA was extracted from the virus by the modified guanidine thiocyanate-phenol method, The titers of the antisense RNA recombinant retrovirus in the supernatant of cell clones were detected by the RT-nested PCR method. At the same time the virus titers were detected by the traditional rat fibroblast magnifying method too. The results detected by the two methods showed that the RT-nested PCR is more specific, sensitive, rapid and simple than the traditional method to detect retrovirus titers.

关 键 词：重组逆转录病毒 反义RNA 逆转录套式PCR 病毒效价 

分 类 号：S852.659.3[农业科学—基础兽医学] Q503[农业科学—兽医学]