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当前位置: 首页 > 文章 > 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 植物病理学报 2011,41 (2) 154-160
Position: Home > Articles > Comparison of different size of probes,labels and detection substrates for diagnosis of Potato spindle tuber viroid by nucleic acid spot hybridization Acta Phytopathologica Sinica 2011,41 (2) 154-160

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

作  者：
吕典秋;刘尚武;邱彩玲;李勇;宿飞飞;王绍鹏;吕文河
单  位：
黑龙江省农业科学院植物脱毒苗木研究所;东北农业大学农学院
关键词：
马铃薯纺锤块茎类病毒;探针;核酸斑点杂交;标记
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。
译  名：
Comparison of different size of probes,labels and detection substrates for diagnosis of Potato spindle tuber viroid by nucleic acid spot hybridization
作  者：
LV Dian-qiu1,2,LIU Shang-wu2,QIU Cai-ling2,LI Yong2,XU Fei-fei2,WANG Shao-peng2,LV Wen-he1(1College of Agronomy,Northeast Agricultural University,Harbin 150030,China;2Virus-free Seedling Research Institute of Heilongjiang Academy of Agricultural Sciences,Supervision and Testing Center for Virus-free Potato Seeds of Ministry of Agriculture,Harbin 150086,China)
关键词：
Potato spindle tuber viroid(PSTVd);probe;nucleic acid spot hybridization(NASH);labeling
摘  要：
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.

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