两种植原体的PCR检测及其体系优化

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1.5 kb的特异片段,所需PCR模板DNA用量为20 ng/μL;在PCR基础上的巢式 PCR扩增出1.2 kb的特异片段,所需PCR模板DNA用量为2 pg/μL.检测灵敏度提高约10 000倍.优化试验表明:25 μL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:25 mM MgCl21.7 μL,2.5 mM dNTP 1.8 μL,5U/μL Taq酶可选用0.2 μL,10mM引物对各0.2 μL,最佳退火温度为45.0℃.

PCR Detection of Two Phytoplasma and Optimization of Reaction System

Using the two general pairs of primers published Phytoplasma from Flavescence dorée 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 25 μL reaction system.Finally the best system was established: 25m M MgCl2 1.7 μL,2.5 mM dNTP 1.8 μL,5 U/μLTaq Enzyme 0.2 μL,10 mM primers 0.2 μL respectively,the best annealing temperature was 45.0℃.