马铃薯病毒检测的研究及其试剂盒的开发-遗传学专业毕业论文.docxVIP

南开大学硕士学位论文摘 南开大学硕士学位论文 摘 要 马铃薯x病毒(PVX)、马铃薯Y病毒(PVY)、马铃薯S病毒(PVS)和 马铃薯卷叶病毒(PLRV)是严重危害我国马铃薯生产的四种I乇NA病毒。目前， 在马铃薯病毒检测中普遍采用的ELISA法复杂费时，灵敏度低，成本高。有文 献报道，可使用传统的RT-PCR方法对马铃薯的叶片和块茎进行病毒检测，但由 于操作复杂且成本高，不能满足生产实践中的应用。 本实验简化了马铃薯茎和叶片组织的RNA提取方法并摸索出一步法 RT-PCR(One-tube RT-PCR)反应体系，从而建立起一套简便有效的马铃薯病毒检 测体系。依据上述四种马铃薯病毒序列的保守区域分别设计引物，采用新建立的 检测体系对染病的马铃薯进行病毒检测，得到与预期相一致的特异扩增片段。将 扩增产物进行克隆、测序，结果与GenBank中登录的病毒序列中相应的核酸序 列基本一致，同源性均达到96％以上。另外，还利用此检测体系对经浓度梯度稀 释的病毒核酸进行检测，以对反应的灵敏度做出初步分析：并用DAS．ELISA法 设计实验，将检测结果与本实验所建立的病毒检测体系的检测结果相对照，得到 了一致的结果。这些结果都表明，改进后的马铃薯病毒检测体系具有快速、可靠、 简便等优点，同时这些工作也为马铃薯块茎的病毒检测等进一步探索奠定了基 础。在此基础之上，本实验又尝试应用一步法多重RT-PCR同时进行多种病毒的 检测，也得到了很好的结果。为了实现对病毒的定量，本实验又采用了TaqMan 实时定量PCR来研究样品中病毒的含量，并将定量检测的结果与通过浓度梯度 稀释进行半定量得到的结果对比，一致性良好。这部分工作也为今后对病毒进行 定量研究开辟了道路。 关键词：马铃薯病毒，RT-PCR，一步法RT-PCR，多熏RT-PCR，TaqMan实时定 量PCR 堕茎奎兰雯主堂竺堡苎—————————!!!LAbstract 堕茎奎兰雯主堂竺堡苎—————————!!!L Abstract Potato virus X fP嘲?Potato virus Y(P嘲．Potato virus S(PyS)and Potato leaf-roll virus(PLR V)are four viruses that lead to reduction ofpotmo output in China． Currently，ELISA has been employed to assess potato viruses，however it is time consuming and not sensitive enough．RT-PCR protocol has been developed to detect potmo virus，but it is laborious and expensive． To develop all effective detection of potato viruses，a simple RNA extraction protocol of potato stems and leaves and one-tube RT-PCR were put forw甜d in this thesis．Four pairs of primers，which designed based on the sequences of the four viruses’RNA，were used in detection for infected samples．The amplified fragments from infected samples were accordant witll expected．Mter cloned and sequencing of amplified fragments，it was resulted that the sequences were almost identical(≥96％ identity)with the viruses sequences reported in GenBank．Viruses’RNA extractions， which diluted in grads，had been detected using the new procedure．Applying DAS—ELISA protocol by comparison，infected samples were detected by both RT-PCR and DAS—ELISA，and got same result．It Can be concluded from above that the new