【作者】 李红霞；

【导师】 谭天伟； 朱水芳；

【作者基本信息】 北京化工大学 ， 生物化工， 2007， 硕士

【摘要】 化学合成的寡核苷酸及其衍生物在聚合酶链式反应（PCR）、生物芯片、基因诊断、分子结构分析及疾病治疗等领域有着广泛的应用，随着生物技术不断地发展和日益广泛的应用，寡核苷酸的需求量也在快速增长，必将拥有更为广泛的应用领域和广阔的市场空间。黄瓜绿斑驳花叶病毒（Cucumber green mottle mosaic virus，CGMMV）是我国重要的对外检疫性有害生物，为烟草花叶病毒属（Tobamovirus）成员，主要侵染葫芦科植物，可导致严重减产甚至绝产。以前我国无此病毒分布，近几年国內从进口种子的葫芦科植物上发现该病毒，它造成了数以千亩计的西瓜等作物绝产和出口退货等重大经济损失。建立该病毒的快速、准确、灵敏的检测和监控技术，是防止该病毒传入国内及扩散流行的关键环节，意义重大。本论文是在前人工作的基础上并结合自己探索性的实验，建立了两种Taqman探针及引物的合成和纯化技术条件，并利用自行合成的Taqman探针及引物在国内首次采用PCR检测方法，从南瓜果实中检测发现CGMMV，建立了南瓜果实中CGMMV的反转录PCR（RT-PCR）和实时荧光PCR（RTF-PCR）检测方法。主要工作如下：采用固相合成方法，使用DNA合成仪自动合成了一对检测CGMMV的引物，采用OPC、PAGE、反相HPLC等不同的纯化方法，对引物的纯化进行了系统地研究，比较其优缺点，建立了引物纯化的实用技术体系。本文应用自行合成的引物，对来自甘肃的疑似染病的南瓜样品进行RT-PCR扩增，成功地得到了阳性扩增片段，经测序和分析，结果表明：扩增片段的大小为660 bp，包含CGMMV的外壳蛋白基因和3′非编码区，与已发表的CGMMV分离物进行核酸序列比对，核苷酸序列同源性为98—99％，在中国首次证明南瓜果实中存在CGMMV。其测序结果同时表明：本实验合成和纯化的引物序列完整、准确，PCR扩增效率高。本文应用DNA自动荧光标示技术，使用DNA合成仪，合成出FAM／TAMRA、FAM／BHQ₂标记的Taqman探针和一对特异性引物，产物采用反相液相色谱法分别纯化，并应用于CGMMV的RTF-PCR检测，首次建立了南瓜果实中CGMMV的RTF-PCR检测方法。多次实验结果证明，自行合成的Taqman探针检测性能良好，其中FAM／BHQ₂探针没有荧光本底，与FAM／TAMRA探针相比，其RTF-PCR检测灵敏度更高。RTF-PCR是目前世界上最快速、灵敏、准确的分子检测技术，引物、探针的纯度和正确性，直接影响检测的准确性和灵敏度。实验结果进一步证明了我们建立的探针合成、纯化体系的高效率和高准确性，为RTF-PCR性能的高效发挥打下了基础。更多还原

【Abstract】 Oligonucleotides and their derivatives have extensive applications in many fields, such as polymerase chain reaction （PCR）, biological DNA chips, gene diagnoses, analysis of molecular structure and treatment of illness. With the rapid development of biotechnology, the demand of Oligonucleotides is increasing. Therefore the products of oligonucleotides have more widespread applications and larger market share.Cucumber green mottle mosaic virus （CGMMV） is an important quarantine plant virus in our country. CGMMV, a member of the Tobamovirus genus, mainly infects the cucurbit species, resulting in the decrease of crop yield or no harvest. Formerly, the virus didn’t existed in China. In recent years, it was detected from cucurbit plants imported to China which led to the death of thousand acres of watermelon and great economic losses for exporting agricultural products. It is significant to establish a rapid, accurate, and sensitive detective technique for prevention of CGMMV from invading and spreading in our country. In this thesis, the synthesis and purification methods of primer and Taqman probes were established and the methods of detecting the CGMMV by RT-PCR and RTF-PCR were set up. The details are as follows:A pair of specific primers had been synthesized by solid phase synthesis method on DNA synthesizer and been purified by OPC, PAGE and RP-HPLC method. The synthesis and purification method were established that we reported here. For the first time, the RT-PCR detection of CGMMV from pumpkin fruit was reported in China. The primers were used for the RT-PCR amplification of infected pumpkin fruit. The expected positive amplified fragments were obtained, sequenced and analyzed. The results showed that fragment contains the coat protein gene and its 3’ noncoding region and is 660nts in size. Comparing with the sequences of other isolators of CGMMV which had been published, the result showed that nucleotides homology was 98-99%.The above data showed first time in China that CGMMV exists in pumpkin fruit, sequences of the primers synthesized and purified in our lab were correct, and they worked efficiently for PCR amplification.FAM/TAMRA, FAM/BHQ₂ Taqman probes and a pair of specific primers were synthesized automatically and purified by RP-HPLC, and they were used for real-time-flurorecent PCR （RTF-PCR） detection of CGMMV. RTF-PCR detection of CGMMV in pumpkin fruit is first reported in the world. It is demonstrated that the sensitivity of RTF-PCR with the FAM/BHQ₂ probe was higher than that of FAM/TAMRA probe due to its low fluorescence background. Up to date, the RTF-PCR is the fastest, the most sensitive and accurate molecular detection method. The qualities of primers and probes have important influences to the sensitivity and accuracy of detection of the method. The results showed that the synthesis and purification system of primers and probes was high efficient and laid the foundation of the high performance of RTF-PCR.更多还原