一种马铃薯病毒RT-PCR诊断新方法

A Novel RT-PCR Protocol for the Detection of Potato Virus

对传统的植物病毒RNA苯酚提取法进行改进;同时设计并优化单管RT-PCR(O ne-tube RT-PCR)方法,使传统的RT-PCR两步反应在一个反应体系中同时进行,依据马铃薯Y病毒和马铃薯S病毒的基因组RNA保守序列设计2套特异性引物,采用上述新建立的检测方法对染病的马铃薯叶片组织进行病毒检测,结果与G eneB ank中报道的这两种马铃薯病毒核苷酸序列进行B last比较,同源性达到96%以上,用这种新型诊断方法对马铃薯病毒核酸进行了浓度检测,实验结果证明,这种新型马铃薯病毒诊断方法具有可靠、快速、简便等特点.

Our research highlights on making a simple nucleic acid extraction protocol of potato stem and leaves, and optimizing the condition of one-tube RT-PCR. Two pairs of primers were designed based on the sequences of the Potato virus Y and Potato virus S, The new procedure of detection was applied with total RNA of the infected potato leaves and stems. The expected fragments were amplifed from the infected smaples. The PCR products were cloned and sequenced. The results showed that the sequences were almost identical C〉96% identity）, compared with the sequences of the viruses＇ RNA reported in GeneBank. Using the new procedure of detection, in addition, we detected the viruses＇ nucleic acid extractions that have been diluted gradually. From the results of above, we can see that the new detection procedure is time consuming and reliable.