不同探针大小、标记物及反应底物对马铃薯类病毒杂交检测的影响  

Comparison of different size of probes,labels and detection substrates for diagnosis of Potato spindle tuber viroid by nucleic acid spot hybridization

作　　者：吕典秋[1,2] 刘尚武[2] 邱彩玲[2] 李勇[2] 宿飞飞[2] 王绍鹏[2] 吕文河[1] 

机构地区：[1]东北农业大学农学院,哈尔滨150030 [2]黑龙江省农业科学院植物脱毒苗木研究所,农业部脱毒马铃薯质量监督检验测试中心,哈尔滨150086

出　　处：《植物病理学报》2011年第2期154-160,共7页Acta Phytopathologica Sinica

基　　金：国家科技部基础研究重大项目(2004ccc02900);黑龙江省攻关重大项目(GA08B102);农业部马铃薯产业创新体系(NYCYTX-15)

摘　　要：灵敏可靠地检测马铃薯纺锤块茎类病毒(Potato spindle tuber viroid,PSTVd)对脱毒种薯的生产具有重要意义。本研究探索了影响核酸杂交检测技术的关键因素。通过基因克隆技术构建了插有PSTVd全长单体、双体及片段的载体。分别以地高辛和同位素为标记物,利用PCR和转录标记技术制备cDNA和RNA探针。比较探针大小、标记物、标记方法、反应底物等对检测灵敏度的影响。结果显示,以地高辛为标记物,利用PCR标记制备的PSTVd双体cDNA探针,在以CDP-Star为底物,通过在柯达X-OMAT BT胶片进行化学发光反应来分析结果的检测灵敏度最高,可以检测到0.05 pg总RNA中的PSTVd,是国外报道检测灵敏度的500倍。利用核酸斑点杂交技术检测PSTVd具有灵敏度高,一次可检测样品数量多等特点,对于大规模PSTVd检测更加方便可行。Sensitive and accurate detection of Potato spindle tuber viroid（PSTVd） is significant for seed potato production.Recombinant DNAs containing full-length monomeric,dimeric and partial-length PSTVd cDNA were constructed,and the various sized PSTVd-cDNAs were labeled by PCR amplification.For comparing the detection sensitivity and specificity of different labeled methods and reaction substrates,the monomeric PSTVd cDNA was also labeled with α-32P-UTP by transcriptional technique.Colorimetric and chemilumine-scent reactions in detection were also compared in N＋-nylon membrane and Kodark film,respectively.The results showed that PSTVd dimeric probe,labeled with DIG and using CDP-Star substrate for chemiluminescent detection,produced the best sensitivity.The detection sensitivity was approximate 0.05 pg PSTVd total RNA,which is 500-fold of the previous published data.This detection system of NASH is high sensitive,specific and feasible for diagnosis of a large number of samples.

关 键 词：马铃薯纺锤块茎类病毒 探针 核酸斑点杂交 标记 

分 类 号：S435.313[农业科学—农业昆虫与害虫防治]