两种植原体的PCR检测及其体系优化  

PCR Detection of Two Phytoplasma and Optimization of Reaction System

作　　者：马春春[1,2] 张祥林[1] 乾义柯[1,2] 王羽中 

机构地区：[1]新疆出入境检验检疫局,乌鲁木齐830063 [2]新疆农业大学农学院,乌鲁木齐830052

出　　处：《新疆农业科学》2009年第3期561-566,共6页Xinjiang Agricultural Sciences

基　　金：国家“十一五”科技支撑计划重大项目(2006BAK10B06)

摘　　要：用已报道的植原体通用引物对葡萄黄化病和枣疯病植原体进行常规PCR及巢式PCR检测,并对PCR反应体系进行优化。结果表明:常规PCR扩增出了1.5kb的特异片段,所需PCR模板DNA用量为20ng/μL;在PCR基础上的巢式PCR扩增出1.2kb的特异片段,所需PCR模板DNA用量为2pg/μL。检测灵敏度提高约10000倍。优化试验表明:25μL反应体系中,MgCl2用量对扩增效果影响最大。最终最佳反应体系确立为:25mM MgCl21.7μL,2.5mM dNTP1.8μL,5U/μL Taq酶可选用0.2μL,10mM引物对各0.2μL,最佳退火温度为45.0℃。Using the two general pairs of primers published Phytoplasma from Flavescence doree and Jujube witches＇ broom were detected by PCR and Nested - PCR and the reaction system was optimized. The results indicated that a phytoplasma - specific 1.5 kb fragment amplified with PCR , the minimal amount of DNA was 20 ng/μL; a phytoplasma- specific 1.5 kb fragment amplified with Nested- PCR, the minimal amount of DNA was 2 pg/μL. The sensitiveity was enhancesed by about 10 000 times. The study of optimization indicated that MgCl2 was found to have distinct effect on amplification in 25μL reaction system. Finally the best system was established： 25m M MgCI2 1.7 μL,2.5 mM dNTP 1.8 μL,5 U/μL Taq Enzyme 0.2 μL, 10 mM primers 0.2 μL respectively, the best annealing temperature was 45.0℃.

关 键 词：植原体 PCR 巢式PCR 体系优化 

分 类 号：S41[农业科学—植物保护]