摘要
以健康昆诺阿藜叶片和受ASGV侵染的昆诺阿藜叶片为试材,利用Trizol试剂提取植物总RNA,将实时荧光RT-PCR体系的主要成分设定梯度,根据每个成分的变化引起的(Rn值和Ct值的差异,探讨苹果茎沟病毒实时荧光RT-PCR技术中反应体系的主要成分对扩增结果的影响。最终确定了RT-PCR反应体系的最佳条件为:25μL体系中,Mg2+浓度为3.5 mM,引物浓度为0.6μM,探针浓度为0.6μM,总RNA含量为20 ng。利用此反应体系进行扩增,△Rn值比未优化时高出一个数量级,表明该体系适合ASGV实时荧光RT-PCR检测分析。
Total RNA of the health leaves and the leaves infected by ASGV was extraction by Trizol reagent. Every factor of real-time fluorescent RT-PCR had different concentrations and its variation changed the result of ARn and Ct. Then the factors affecting the real-time fluorescent RT-PCR results of ASGV were studied. The results showed that the best reaction system was: 3.5 mM of Mg^2+ , 0. 6μM of either primer,0. 6 μM probe, and 20 ng total RNA in 25 μL reaction mixture. ARn was ten times as high using above optimal real-time fluorescent RT-PCR system than original system. This indicated this system was suitable for detection of ASGV.
出处
《北方园艺》
CAS
北大核心
2009年第3期8-11,共4页
Northern Horticulture
基金
福建省自然科学基金资助项目(2006J0291)