發(fā)布時(shí)間：2018-03-18 03:35

  本文選題：鼠疫菌　切入點(diǎn)：PNA　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2006年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：利用特異的肽核酸(Peptide Nucleic Acid，PNA)探針、鏈霉親和素包被的磁性納米顆粒(磁珠)和Cy5納米顆粒，結(jié)合熒光掃描技術(shù)，建立一種特異、快速、準(zhǔn)確的檢測鼠疫菌的方法。 方法：針對鼠疫菌pMT1質(zhì)粒上的caf1基因設(shè)計(jì)并合成一對特異PNA探針，經(jīng)生物素標(biāo)記后，分別與鏈霉親和素包被的磁珠或Cy5納米顆粒結(jié)合；探針與待測鼠疫菌的DNA雜交后，磁性納米顆粒牢固連接靶核酸-探針結(jié)合體，用磁分離架收集后即可得純化的特異核酸分子，經(jīng)變性處理后對Cy5熒光納米顆粒進(jìn)行熒光掃描檢測。本研究探討了多個(gè)實(shí)驗(yàn)因素對測定的影響，并進(jìn)行了特異性和靈敏度檢測。此外，本研究將PNA探針與DNA探針、磁性納米顆粒與市售磁珠、Cy5熒光納米顆粒標(biāo)記的探針與單個(gè)Cy5分子標(biāo)記的探針進(jìn)行比較，從而驗(yàn)證PNA探針、磁性納米顆粒及Cy5熒光納米顆粒在核酸檢測技術(shù)中的優(yōu)勢。 結(jié)果：利用Expedite 8909核酸合成系統(tǒng)合成了PNA探針，建立并優(yōu)化了利用PNA探針檢測鼠疫菌的方法，得到較好的線性關(guān)系(r=0.9914)；檢測的靈敏度為O.9μg／mL(待測DNA)。通過多項(xiàng)比較，驗(yàn)證了PNA探針、磁性納米顆粒及Cy5熒光納米顆粒在核酸檢測技術(shù)中的優(yōu)勢。 結(jié)論：PNA探針的結(jié)構(gòu)特點(diǎn)決定了其具有卓越的雜交親和性能和特異識別能力，從而使本研究顯示出較高的特異性和靈敏度，，具有較好的應(yīng)用前景；磁性納米顆粒的應(yīng)用可簡便且有效地將雜交復(fù)合體與未雜交核酸或過量的檢測探針分離，大大降低非特異核酸分子的干擾，提高了檢測靈敏度；Cy5熒光納米顆粒具有的光穩(wěn)定性和信號放大作用特點(diǎn)，使基于熒光納米顆粒的熒光分析方法的靈敏度較傳統(tǒng)方法顯著提高。本研究建立的分析方法檢測的靈敏度和特異性較高，能夠靈敏、特異、穩(wěn)定地對鼠疫菌進(jìn)行定量檢測，為鼠疫監(jiān)控、診斷、應(yīng)對緊急疫情的發(fā)生、動態(tài)的疫源地疫情監(jiān)測提供了快速檢測鑒定的有力手段。
[Abstract]:Objective: to establish a specific, rapid and accurate method for the detection of Yersinia pestis by using a specific peptide nucleic acid (PNA) probe, magnetic beads and Cy5 nanoparticles coated with streptavidin. Methods: a pair of specific PNA probes were designed and synthesized for the caf1 gene on the pMT1 plasmid of Yersinia pestis, which were labeled with biotin and bound to the magnetic beads or Cy5 nanoparticles coated with streptavidin, respectively, and hybridized with the DNA of Yersinia pestis. The magnetic nanoparticles are firmly connected to the target nucleic acid-probe, and the purified specific nucleic acid molecules can be obtained by magnetic separators. Cy5 fluorescent nanoparticles were detected by fluorescence scanning after denaturation. In this study, the effects of several experimental factors on the determination were investigated, and the specificity and sensitivity were detected. In addition, the PNA probe and the DNA probe were used in this study. The advantages of PNA probe, magnetic nanoparticles and Cy5 fluorescent nanoparticles in nucleic acid detection were verified by comparing the probe labeled with magnetic nanoparticles Cy5 fluorescent nanoparticles and those labeled by single Cy5 molecule in order to verify the advantages of PNA probes, magnetic nanoparticles and Cy5 fluorescent nanoparticles in nucleic acid detection. Results: the PNA probe was synthesized by using Expedite 8909 nucleic acid synthesis system, and the method of detecting Yersinia pestis with PNA probe was established and optimized. A good linear relationship was obtained, and the sensitivity of detection was 0. 9 渭 g / mL. Advantages of magnetic nanoparticles and Cy5 fluorescent nanoparticles in nucleic acid detection. Conclusion the structure of the probe determines that it has excellent hybridization affinity and specific recognition ability, which makes this study show high specificity and sensitivity, and has a good prospect of application. The magnetic nanoparticles can be used to separate the hybrid complex from non-hybrid nucleic acid or excessive detection probe, which greatly reduces the interference of non-specific nucleic acid molecules. The detection sensitivity of Cy5 fluorescent nanoparticles is improved, which has the characteristics of optical stability and signal amplification. The sensitivity of the fluorescent analysis method based on fluorescent nanoparticles was significantly higher than that of the traditional method. The sensitivity and specificity of the analytical method established in this study were higher than that of the traditional method. The method was able to detect Yersinia pestis quantitatively with sensitivity, specificity and stability. It provides a powerful means for rapid detection and identification of plague surveillance, diagnosis, emergency outbreak and dynamic foci surveillance.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R378

【參考文獻(xiàn)】

相關(guān)期刊論文 前8條

1 熱娜·吐爾地,徐秉臣,阿扎提·熱合木,孫石,雷剛;鼠疫F1McAb酶免疫染色法快速檢驗(yàn)鼠疫菌[J];地方病通報(bào);2003年01期

2 宋延富;鼠疫以非典型形式在自然界長期保存的研究進(jìn)展[J];中國地方病防治雜志;1995年02期

3 李書寶,戴繪;世界鼠疫疫情態(tài)勢及研究進(jìn)展[J];中國地方病防治雜志;2000年01期

4 浦清江,張春華,叢顯斌,趙志海;鼠疫放射免疫沉淀試驗(yàn)在鼠疫監(jiān)測中的應(yīng)用及效果評價(jià)[J];中國地方病防治雜志;2002年05期

5 王曉春,高崇華;關(guān)于我國鼠疫疫情趨勢的思考[J];中國地方病防治雜志;2003年04期

6 黃海,馬文麗,董興齊,張寶,吳清華,鄭文嶺;基因芯片用于鼠疫快速診斷的初步研究[J];第一軍醫(yī)大學(xué)學(xué)報(bào);2004年01期

7 黃惠杰,翟俊輝,任冰強(qiáng),楊瑞馥,趙永凱,程兆谷,杜龍龍,路敦武;光纖倏逝波生物傳感器及其應(yīng)用[J];光學(xué)學(xué)報(bào);2003年04期

8 于學(xué)東,裴德翠,宋亞軍,郭兆彪,王津,李永勤,楊瑞馥;多重PCR快速鑒定鼠疫耶爾森氏菌[J];中國人獸共患病雜志;2003年03期

相關(guān)博士學(xué)位論文 前1條

1 何曉曉;生物親和性核殼納米顆粒研究及其在生物/醫(yī)學(xué)中的應(yīng)用[D];湖南大學(xué);2003年

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