西伯利亚百合病毒RNA提取及RT-PCR方法的比较与优化  

Comparison and Optimization of the Methods on RNA Extraction of Three Viruses and RT-PCR in Siberia Lilium

作　　者：赵庆芳[1] 韩静[1] 马平霞[1] 丁文龙[1] 李巧峡[1] 

机构地区：[1]西北师范大学生命科学学院,甘肃兰州730070

出　　处：《北方园艺》2008年第7期201-204,共4页Northern Horticulture

基　　金：甘肃省科技攻关计划资助项目(2GS042-A41-001-11);兰州市科技局资助项目(06-2-19)

摘　　要：在对感染西伯利亚百合的CMV、LSV、LmoV 3种病毒的检测上,对4种常见的RNA提取方法及影响RT-PCR体系的因素进行了比较研究。改良优化了的RNA提取方法,不需用DEPC水处理和高温烘器皿,省去液氮研磨过程,采用该法从百合叶片中提取总RNA,整个过程及后继反应均不需使用RNasin,降低了成本,同时提取的RNA质量高,适于后期RT-PCR的检测,更适合于百合幼嫩叶片和试管苗RNA的提取。运用一步法,建立了CMV、LSV、LMoV 3种病毒的RT-PCR优化体系,最优反应体系为:M-MLV 5×Reaction Buffer 1μL1、0×PCR buffer0.5μL2、.5 mM dNTPs(each)0.8μL1、0 pmol/μL下游引物0.2μL1、0 pmol/μL上游引物0.2μL、M-MLVRT 200 U/μL 0.1μL2、.5 U/μL Taq polymerase 0.1μL;0.4μL感病材料RNA提取液,剩余部分用无菌双蒸水补足,使反应体系达到10μL。CmV、LSV and LMoV are three viruses that damage lily seriously. The application of molecular biology method of RT-PCR technology was to exam the virus of Siberia Lilium. The methods of RNA extraction and RT-PCR were compared and optimized in Siberia Lilium. In this research, the improved method of RNA extraction that had the following merits： it neither needs to dry the household utensils with the DEPC water treatment and high temperature, nor needs the fluid nitrogen to grind the material, which save a lot of preparatory work. The application of this method to extract RNA from the lily leaves without using RNasin in the whole process and its successive reaction could reduce the cost, save resources and time, and was more suitable for the extraction of the young tender lily leaves and test tube seedling RNA. What＇s more, the extracted RNA was suitable for the later RT-PCR examination. The optimization of the RT -PCR examilmtion system of cmV,LSV,and LmoV were established by using the one-stage process of RT-PCR . The optimized reacting system was M-MLV 5 Reaction Buffer 1μL.10×PCR buffer 0.5μL、2.5 mM dNTPs（each）0.8μL. 10 pmol/μL Down printer 0.2μL,10 pmol/μL Up primer 0.2μL.M-MLVRT 200 U/μL 0.1/μL.2.5 U/μL Thq poly-merase 0.1/μL; 0.4 /μL Infected materials such as extracted liquid RNA. Surplus part can be supplemented by aseptic double steam water to enables the reacting system to reach 10/μL.

关 键 词：西伯利亚百合 RNA提取 病毒检测RT-PCR 

分 类 号：S682.29[农业科学—观赏园艺]