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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 reliable detection of Potato spindle tuber viroid (PSTVd) is of great importance to the production of virus-free seed potato. This study explored the key factors affecting the detection of nucleic acid hybridization. Through the gene cloning technique, a full-length insert of PSTVd monomers, diabodies and fragments were constructed. Using digoxigenin and isotope as markers respectively, cDNA and RNA probes were prepared by PCR and transcriptional labeling. The effect of probe size, label, labeling method and reaction substrate on the detection sensitivity was compared. The results showed that the detection sensitivity of the PSTVd catalase cDNA prepared by PCR using digoxigenin as a marker and CDP-Star as a substrate by chemiluminescence reaction on Kodak X-OMAT BT film The highest, can detect 0.05 pg of total RNA in the PSTVd, is reported sensitivity of foreign reports 500 times. The detection of PSTVd by nucleic acid dot blotting has the characteristics of high sensitivity and large number of detectable samples at one time. It is more convenient and feasible for large-scale detection of PSTVd.