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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 pairs of primers containing BamHI overlop were designed at the BamHI site (87-92 nt) in the genome of potato spindle tuber viroid (PSTVd). The full-length PSTVd obtained by PCR amplification was inserted into the pGM-T vector, respectively. Through the BamH I site, two PSTVd monomers were ligated to construct a PSTVd diabody vector. Using this vector as a template, a highly sensitive and specialized PSTD double-labeled cDNA digoxigenin-labeled probe was prepared by PCR. CDP-Star as the reaction substrate for chemiluminescence reaction interpretation, the establishment of PSTVd efficient hybridization detection system. The system can detect potato tuber, leaves, buds and other tissue samples with 0.05pg detection sensitivity. A total of 67 field samples collected from laboratory and resource materials stored in the laboratory were tested. CDNA hybridization of nucleic acid spot hybridization (NASH), reverse-transcriptional The detection rate of positive samples by RT-PCR and return-polyacrylamide gel electrophoresis (R-PAGE) showed that the positive rate of NASH positive samples was 67.7% , Consistent with RT-PCR, higher than the positive detection rate of R-PAGE technology (53.7%).