苹果茎痘病毒RT-PR检测体系的建立.doc

苹果茎痘病毒RT-PCR检测体系的建立 学生：钟敏 指导教师：王亚南 （河北农业大学植物保护学院 河北保定 071000） 摘要：苹果为世界四大水果之一，河北省是我国渤海湾苹果产区的重要省份之一，苹果种植面积位居全省果品面积首位。苹果病毒病是苹果生产的大敌，目前，河北省苹果病毒病的发生情况还不清楚，而且由于苹果组织中含多糖多酚、苹果病毒含量低，苹果病毒检测体系还不成熟。本研究针对以上问题，对苹果茎痘病毒（Apple stem pitting virus，ASPV）扩增引物进行筛选并对RT-PCR检测体系及程序进行优化。结果表明：ASPV(Z)、ASPV(Xin)、2002ASPV、ASPV(Y)几对扩增引物中，2002ASPV能检测出特异性条带且灵敏度高，适合田间检测；通过优化RT-PCR体系得出，在25μL 总体系中，最佳cDNA模板量为0.25 μL、3’与5’端引物1.0 pmol、dNTPs 2.5 μL；通过对PCR程序的优化得出，rTaq DNA聚合酶最适用量为1.50 U、循环次数35个循环、最适退火温度为57.6℃。最后通过对RT-PCR产物进行测序，确定扩增条带与目的片段DNA序列相似性高，与已发表的序列相似性达82%-90% 。 关键词：苹果茎痘病毒；引物筛选；RT-PCR优化 Establishment of RT-PCR detection system for Apple stem pitting virus Candidate： ZHONG Min Advisor： WANG Ya-nan (College of plant protection, Agricultural University of Hebei, Baoding, Hebei 071001, China) Abstract： Apple is one of the important fruits in the world. Hebei province is one of the important apples planting area in China Bohai Bay region. Apple virus disease is the enemy of apple production in Hebei province. At present, the infection of apple virus is not clear, but due to the apple organization containing polysaccharide, the virus of apple content is low and apple virus detection system is not mature. This study aiming at the above problems to select primers of Apple stem pitting virus and optimize RT-PCR system and program. The results showed that, in the primers of ASPV (Z), ASPV (Xin), 2002ASPV, ASPV (Y) , the 2002ASPV could detect specific fragment with high sensitivity and which is suitable for field. detection; The result of RT-PCR system component optimizing was as follows: cDNA template was 0.25 μL, 3 and 5 primers 1.0 pmol, dNTP 2.5 mM, which could show the brightest and specific fragment; The PCR program is optimized, the result was rTaq 2.50U, cycle number 35. The most suitable annealing temperature was 57.6℃; The RT-PCR products were sequenced, the fragment size consistent with published DNA sequence with 82%-90%similarity to the published ASPV sequence. Key words: ASPV; Primers screeni