马铃薯病毒检测-遗传学专业毕业论文.docxVIP

南开大学硕士学位论文 南开大学硕士学位论文 摘要 摘要 马铃薯x病毒(PVX)、马铃薯Y病毒(PVY)、马铃薯S病毒(PVS)、 马铃薯卷叶病毒(PLRV)是严重危害我国马铃薯产量的四种病毒，目前，普遍 采用ELISA法检测马铃薯病毒，但它存在费时、成本高、灵敏度不足的缺点， 国内外有文献报道，用传统的RT．PCR方法对马铃薯的叶片和块茎进行病毒检 测，但由于操作步骤复杂，检测成本较高，不适于在生产实践中应用。 本实验室研究突出在简化马铃薯茎和叶片组织的核酸提取方法以及优化 RT．PCR一步反应体系(One—tube RT．PCR)，从而建立起一套完整而有效的马铃 薯病毒检测体系。我们依据上述4种马铃薯病毒的RNA序列保守区域分别设计 特异性引物，采用新建立的检测体系对染病的马铃薯茎和叶片组织进行病毒检 测，得到与预期一致的特异性病毒扩增片段。将扩增产物进行克隆、测序，结 果与GeneBank中报道的病毒序列对应部分的核苷酸序列基本一致，同源性达 到96％以上，除此以外，我们用此检测体系对病毒核酸进行浓度梯度检测，用 DAS--ELISA法设计对照实验，以上实验结果表明，改进后的马铃薯病毒检测 体系具有快速、可靠、简便等特点，同时这些工作也为马铃薯块茎的病毒检测 以及多重PCR体系设计等进一步探索奠定了基础。 关键词：马铃薯病毒RT．PCR DAS--ELISA核酸提取One—tube RT-PCR 壹堑奎堂堡主兰垡笙塞 壹堑奎堂堡主兰垡笙塞 一一一————塑 Abstract Potato virus X PVX；Potato vf，us E PVY；Potato virus S，PVS；and Potato leaf- roll virus，PLRV are four viruses that result in decreased yield of potato in china． Currently ELISA has been employed to aSSeSS potato viruses．However，the method is time consuming and isn’t sensitive enough．RT—PCR protocol has been developed to detect potato virus．However it is laborious and expensive． To develop a procedure for effective detection of potato viruses，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，Four pairs of primers were designed baSed on the sequences of the four viruses’RNA．The new procedure of detection was applied with total RNA of the infected potato leaves and stems．The expected fragments were amplified from the infected samples，but not from healthy samples．The PCR products were cloned and sequenced．The results showed that the sequences were almost identical(96％identity)，compared with the sequences of the viruses’RNA reported in GeneBank．Using the new procedure of detection，we detected the viruses’nucleic acid extractions that have been diluted gradually． Applying DAS--ELISA protocol as control，we also detected the infected samples from lab and fields．From the results of above，we can see that the new‘detection procedure is time c