马铃薯类病毒cDNA双体探针的研制及其在检测上的应用

Development of cDNA Dimeric Probes and Its Application in Diagnosis of Potato spindle tuber viroid

在马铃薯纺锤块茎类病毒（Potato spindle tuber viroid,PSTVd）基因组中BamHⅠ位点处（87~92nt）,设计两对含有BamHⅠ重叠区（overlop）的引物。将PCR扩增获得的全长PSTVd分别插入到pGM-T载体中。通过BamHⅠ位点,将两个PSTVd单体连接,构建了PSTVd双体载体。以此载体为模板,通过PCR标记技术,制备了高灵敏高专化的PSTVd双体cDNA地高辛标记探针。以CDP-Star为反应底物进行化学发光反应结果判读,建立了PSTVd的高效杂交检测体系。该体系可以对马铃薯薯块、叶片、芽等不同组织样品进行检测,检测灵敏度达到0.05pg。通过对67份田间采集的样品和实验室保存的资源材料进行检测,比较了cDNA双体探针核酸斑点杂交技术（nucleic acid spot hybridization,NASH）、反转录聚合酶链式反应（reverse-transcriptional polymerase chain reaction,RT-PCR）和往返-聚丙烯酰胺凝胶电泳（return-polyacrylamide gel electrophoresis,R-PAGE）检测技术的阳性样品检出率,结果显示,NASH技术阳性样品检出率为67.7%,与RT-PCR相一致,高于R-PAGE技术的阳性检测率（53.7%）。

Two PCR amplifying full-length PSTVd （Potato spindle tuber viroid） with the BamHI （87 - 92 nt） overlop were linked to pGM-T to construct the recombinant vector containing dimeric PSTVd cDNA. Highly sensitive and specific dimeric probe labeled with digoxin was produced with PCR amplification. An efficient hybridization system was established by with the substrate of CDP-star in this study. The minimum concentration of PSTVd detected by this probe was 0.05 pg. The samples prepared from leaves, tubers and sprouts could be detected successfully. Compared the positive sample percentage detected from potato fields and conserved potato resource of plantlets in vitro with RT-PCR （ reverse-transcriptional polymerase chain reaction） and R-PAGE （returu-polyacrylamide gel electrophoresis）, NASH （nucleic acid spot hybridization） could give the same positive sample percentage as RT-PCR did （67.7%） , but was higher than R-PAGE （53.7%）.