库尔勒香梨的苹果褪绿叶斑病毒cDNA探针检测技术研究

Studies on cDNA Probe Detection Technology of Apple Chlorotic Leaf Spot Virus in Korla Pear

以库尔勒香梨叶片和皮层为材料,对两种植物总RNA提取方法进行了比较研究,从中筛选出了适合库尔勒香梨的提取方法;并利用RT-PCR反应体系成功合成了生物素标记的cDNA探针,在此基础上,对影响杂交灵敏度的主要因素进行了实验研究。结果表明,当探针浓度为400ng/mL,甲酰胺浓度为45%,42℃杂交反应6h,可获得最高灵敏度。Tween20的封闭效果比牛血清白蛋白好。对不同组织和不同提取方法提取的总RNA进行了杂交检测,并对库尔勒地区香梨带毒情况进行了检测。结果表明,不论幼叶、老叶、皮层及冷冻叶片均可出现杂交信号,两种提取方法提取的总RNA也都出现了杂交信号,库尔勒香梨样品有45%的带苹果褪绿叶斑病毒。

Using Korla pear leaves and cortexes as materials, we extracted total RNA by two methods. The methods good for Korla pear were screened out; and also Biotin labeled cDNA Probe was produced with RT-PCR. Then main parameters on hybridization were optimized. The highest sensitivity was obtained at probe concentration 400 ng/ml, 45%formamide and hybridization at 42℃ for 6 hours. Tween20 is better blocking agent than Albumin Bovine. Hybridization signals were got from the total RNA of fresh leaves and old leaves and cortexes and freezing leaves extracted by two methods,45% Korla pear trees are infected with ACLSV.