本文關(guān)鍵詞：炭疽芽孢桿菌特征基因恒溫?cái)U(kuò)增檢測方法的研究　出處：《中國科學(xué)院研究生院（武漢病毒研究所）》2007年博士論文　論文類型：學(xué)位論文

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【摘要】： 炭疽是由炭疽芽孢桿菌引起的嚴(yán)重急性傳染病，同時(shí)由于炭疽芽孢在環(huán)境中的抵抗力極強(qiáng)，是潛在的生物戰(zhàn)劑之一，因此研究炭疽芽孢桿菌的檢測對于臨床診斷炭疽和防范生物恐怖威脅具有重要的意義。針對以上需求，本研究將恒溫?cái)U(kuò)增技術(shù)應(yīng)用于炭疽芽孢桿菌的檢測，發(fā)展了炭疽芽孢桿菌的恒溫?cái)U(kuò)增檢測方法，根據(jù)擴(kuò)增技術(shù)的不同，分為環(huán)介導(dǎo)的恒溫核酸擴(kuò)增技術(shù)(loop-mediated isothermal amplification，LAMP)和鏈置換擴(kuò)增技術(shù)(strand displacement amplification，SDA)兩種。 炭疽芽孢桿菌的LAMP檢測方法選擇Ba813序列、pag基因和capB基因?yàn)闄z測目標(biāo)，分別代表炭疽芽孢桿菌的基因組和兩個(gè)毒力編碼質(zhì)粒pXO1和pXO2。以炭疽芽孢桿菌A16純培養(yǎng)物為檢測對象，LAMP方法的檢測靈敏度為10個(gè)芽孢，比多重PCR靈敏度高一個(gè)數(shù)量級。通過對LAMP擴(kuò)增產(chǎn)物酶切及46株細(xì)菌的LAMP擴(kuò)增試驗(yàn)表明該方法檢測炭疽芽孢桿菌具有較高的特異性。隨后以混有炭疽芽孢的粉末為模擬實(shí)際樣品初步評價(jià)了LAMP方法在實(shí)際檢測中的應(yīng)用前景。以上試驗(yàn)均采用凝膠電泳方法檢測LAMP擴(kuò)增產(chǎn)物，本研究也嘗試用三種不同的熒光染料EvaGreen~(TM)、SYBR~(?) Gold以及EB分別檢測Ba813序列、pag基因和capB基因LAMP擴(kuò)增產(chǎn)物，發(fā)展炭疽芽孢桿菌多熒光共檢測LAMP方法，，使LAMP方法的結(jié)果判斷更加簡單、直觀。 炭疽芽孢桿菌的SDA檢測方法選擇pag基因?yàn)闄z測目標(biāo)，以凝膠電泳方法檢測SDA擴(kuò)增產(chǎn)物，初步評價(jià)了該方法的靈敏度和特異性。由于SDA擴(kuò)增產(chǎn)物在凝膠電泳檢測時(shí)經(jīng)常有拖尾現(xiàn)象，目前SDA技術(shù)的常用檢測方法是熒光偏振和FRET技術(shù)，其信號檢測依賴昂貴的熒光檢測儀器。為此，本研究發(fā)展了一種新的SDA-ELISA檢測方法，通過酶學(xué)顯色使SDA產(chǎn)物檢測更加簡單直觀。采用優(yōu)化的反應(yīng)條件，該方法檢測炭疽芽孢桿菌的特異性高，檢測靈敏度為10個(gè)芽孢，比對應(yīng)的凝膠電泳方法高10倍。 本研究發(fā)展的兩種炭疽芽孢桿菌的恒溫?cái)U(kuò)增檢測方法具有較高的靈敏度和特異性且對儀器依賴程度低，具有良好的應(yīng)用前景，該方法的發(fā)展和應(yīng)用為炭疽芽孢桿菌的檢測提供了新的手段。
[Abstract]:Anthrax is caused by Bacillus anthracis severe acute infectious disease, and because the resistance of Bacillus anthracis in the environment is extremely strong, is one of the potential biological warfare agents, so the detection of Bacillus anthracis has important significance for clinical diagnosis and prevention of anthrax bioterrorism threat. According to the above requirements, this study will detect the amplification the technology applied to Bacillus anthracis, Bacillus anthracis developed isothermal amplification methods, according to different amplification technology, divided into a loop mediated isothermal nucleic acid amplification technology (loop-mediated isothermal amplification, LAMP) and strand displacement amplification (strand displacement, amplification, SDA) two.
LAMP method for detection of Bacillus anthracis Ba813 sequences, PAG gene and capB gene as the target detection, representing the Bacillus anthracis genome and two virulence plasmid pXO1 encoding and pXO2. in Bacillus anthracis A16 pure culture method for object detection, LAMP detection sensitivity was 10 spores, ratio of multiple PCR sensitivity an order of magnitude higher. The LAMP products were digested and 46 strains of bacteria were amplified by LAMP test showed that the specificity for detection of Bacillus anthracis is higher. Then mixed with anthrax spores powder application to simulate the actual sample evaluation of the LAMP method in the actual testing. These tests were using gel electrophoresis method for detection of LAMP amplification products, this study tries to use three different fluorescent dye EvaGreen~ (TM), SYBR~ (?) Gold and EB were used to detect the Ba813 sequence of PAG gene and capB gene LAMP The LAMP method of multi fluorescence detection of Bacillus anthracis was developed, which made the results of LAMP method more simple and intuitionistic.
SDA method for detection of Bacillus anthracis PAG gene as the target detection, PCR products by gel electrophoresis method for detection of SDA, a preliminary evaluation of the sensitivity and specificity of this method. The SDA amplification products in gel electrophoresis often tailing phenomenon, the current SDA technology commonly used detection methods of fluorescence polarization and FRET technology, the signal detection on fluorescence detection instrument is expensive. Therefore, this study developed a new method for determination of SDA-ELISA by enzymatic colorimetric detection of SDA product to make more simple and intuitive. The optimized reaction conditions, the specificity of the method for detection of Bacillus anthracis, the detection sensitivity is 10 to 10 times higher than the spores. Gel electrophoresis method corresponding to.
The two isothermal amplification detection methods of Bacillus anthracis developed in this study have high sensitivity and specificity, and have low potential for instrument dependence, which has good application prospects. The development and application of this method provide a new way for the detection of Bacillus anthracis.

【學(xué)位授予單位】：中國科學(xué)院研究生院（武漢病毒研究所）
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R378

【引證文獻(xiàn)】

相關(guān)期刊論文 前2條

1 王紀(jì)東;王小慧;李媛;張娟琨;詹林盛;;切刻內(nèi)切酶介導(dǎo)恒溫?cái)U(kuò)增技術(shù)條件優(yōu)化[J];軍事醫(yī)學(xué);2012年01期

2 秦勝利;王建廣;;溶藻弧菌的依賴于核酸序列恒溫?cái)U(kuò)增檢測方法的建立[J];青島科技大學(xué)學(xué)報(bào)(自然科學(xué)版);2012年01期

相關(guān)碩士學(xué)位論文 前3條

1 曹仁祺;日本血吸蟲病環(huán)介導(dǎo)等溫?cái)U(kuò)增診斷方法的建立和初步應(yīng)用[D];華中農(nóng)業(yè)大學(xué);2010年

2 馮瑜菲;豬水腫病大腸桿菌毒力因子雞卵黃抗體制備及Stx2e基因LAMP方法建立[D];東北農(nóng)業(yè)大學(xué);2011年

3 杜琳琳;南方水稻黑條矮縮病毒分子檢測方法的研究[D];南京農(nóng)業(yè)大學(xué);2012年

本文編號：1422439