超分枝滾環(huán)擴(kuò)增技術(shù)應(yīng)用于幾種海洋產(chǎn)毒微藻的檢測(cè)
發(fā)布時(shí)間：2019-02-17 13:09
【摘要】：全世界每年因赤潮的爆發(fā)消耗了大量的人力、物力和財(cái)力,尤其是產(chǎn)毒藻種引發(fā)的赤潮,其破壞力對(duì)水產(chǎn)養(yǎng)殖業(yè)和海洋生態(tài)環(huán)境均是毀滅性的,因此建立高特異性、高靈敏度的赤潮藻檢測(cè)方法非常必要。本研究以強(qiáng)壯前溝藻(Amphidinium carterae)、赤潮異彎藻(Heterosigma akashiwo)、米氏凱倫藻(Karenia mikimotoi)三種產(chǎn)毒藻種為目標(biāo)藻,探討了超分枝滾環(huán)擴(kuò)增(hyper-branched rolling circle amplification,HRCA)技術(shù)在海洋產(chǎn)毒藻檢測(cè)中的應(yīng)用。(1)以強(qiáng)壯前溝藻為研究對(duì)象,通過(guò)核糖體大亞基(ribosomal large subunit,LSU)rDNA序列的克隆及測(cè)序,將測(cè)序結(jié)果與相關(guān)序列經(jīng)比對(duì)分析獲得種特異性序列,并根據(jù)鎖式探針(padlock probe,PLP)的設(shè)計(jì)原則設(shè)計(jì)特異性的PLP,建立強(qiáng)壯前溝藻HRCA檢測(cè)體系,對(duì)檢測(cè)體系進(jìn)行優(yōu)化、特異性驗(yàn)證和靈敏度測(cè)試,并最終通過(guò)模擬自然水樣和自然水樣對(duì)建立的方法進(jìn)行了評(píng)價(jià)。結(jié)果表明:HRCA反應(yīng)的最佳連接溫度和擴(kuò)增溫度分別是59°C和61°C,并且隨著時(shí)間的延長(zhǎng)產(chǎn)量迅速增加;建立的強(qiáng)壯前溝藻HRCA體系具有極高的特異性和靈敏度,質(zhì)粒檢測(cè)靈敏度為2 fg,細(xì)胞粗提液檢測(cè)極限為1 cells;HRCA體系可以對(duì)模擬自然水樣和自然水樣進(jìn)行有效檢測(cè)。(2)以強(qiáng)壯前溝藻為目標(biāo)藻,探討了將HRCA擴(kuò)增技術(shù)與熒光定量(quantitative real-time PCR,RT-qPCR)技術(shù)相結(jié)合用于目標(biāo)藻的定量檢測(cè)方法,分別建立了強(qiáng)壯前溝藻的熒光定量-超分枝滾環(huán)擴(kuò)增(quantitative real-time hyper-branched rolling circle amplification,RT-qHRCA)和RT-qPCR檢測(cè)體系,對(duì)兩種擴(kuò)增體系進(jìn)行了特異性和靈敏度測(cè)試,并最終通過(guò)模擬自然水樣對(duì)兩種擴(kuò)增方法進(jìn)行了評(píng)價(jià)。結(jié)果表明:RT-qHRCA體系可以檢測(cè)到1.4 cells,而RT-qPCR體系只能檢測(cè)到7.5 cells,并且RT-qHRCA體系可以對(duì)模擬自然水樣進(jìn)行有效檢測(cè)。(3)以赤潮異彎藻為目標(biāo)藻,對(duì)其LSU rDNA進(jìn)行克隆測(cè)序,并以其為靶序列,分別根據(jù)PLP設(shè)計(jì)原則手工設(shè)計(jì)HRCA擴(kuò)增所需的PLP和采用在線設(shè)計(jì)軟件Primerexplore設(shè)計(jì)環(huán)介導(dǎo)恒溫?cái)U(kuò)增(loop-mediated isothermal amplification,LAMP)所需引物,建立了赤潮異彎藻的HRCA和LAMP檢測(cè)體系,優(yōu)化后對(duì)兩種體系進(jìn)行了特異性和靈敏度測(cè)試,并最終通過(guò)模擬自然水樣對(duì)兩種擴(kuò)增方法進(jìn)行了應(yīng)用評(píng)價(jià)。結(jié)果表明:本實(shí)驗(yàn)建立的HRCA體系的特異性要高于LAMP擴(kuò)增體系;兩種擴(kuò)增方法的質(zhì)粒檢測(cè)靈敏度可達(dá)20 fg,細(xì)胞檢測(cè)極限可達(dá)10 cells;并且兩種體系均不受非目標(biāo)基因組的影響。(4)以米氏凱倫藻為目標(biāo)藻,對(duì)其LSU rDNA進(jìn)行克隆測(cè)序,并以其為靶序列,分別根據(jù)PLP設(shè)計(jì)原則設(shè)計(jì)HRCA和雙切口超分枝滾環(huán)擴(kuò)增(double nick hyper-branched rolling circle amplification,dHRCA)所需的PLP,建立了米氏凱倫藻HRCA和dHRCA檢測(cè)體系,對(duì)檢測(cè)體系進(jìn)行了優(yōu)化、特異性驗(yàn)證和靈敏度測(cè)試,并最終通過(guò)模擬自然水樣對(duì)建立的方法進(jìn)行了評(píng)價(jià)。結(jié)果表明:HRCA和dHRCA擴(kuò)增體系均具有極高的特異性;HRCA體系靈敏度要高于dHRCA體系。
[Abstract]:The outbreak of red tide consumes a great deal of human, material and financial resources every year in the world, especially the red tide caused by poisonous algae. Its destructive power is destructive to aquaculture and marine ecological environment, so it has a high specificity. It is necessary to detect red tide algae with high sensitivity. In this study, three toxic algae species of (Amphidinium carterae), (Heterosigma akashiwo), (Heterosigma akashiwo), were used as target algae, and hyper-branched rolling circle amplification, amplification (hyper-branched rolling circle amplification,) was studied. The application of HRCA technique in the detection of marine toxin producing algae. (1) the cloning and sequencing of rDNA sequence of (ribosomal large subunit,LSU, a large ribosomal subunit, was carried out on the basis of DNA sequencing. The result of sequencing was compared with the relative sequence to obtain the specific sequence. According to the design principle of locked probe (padlock probe,PLP), the specific PLP, was designed to establish the detection system of HRCA of Euglans roxburghii, and the detection system was optimized. The method was evaluated by simulating the natural water sample and the natural water sample. The results showed that the optimum joining temperature and amplification temperature of HRCA reaction were 59 擄C and 61 擄C, respectively, and the yield increased rapidly with the extension of time. The established HRCA system has high specificity and sensitivity. The sensitivity of plasmid detection is 1 cells;. The detection limit of the crude extract of 2 fg, cells is 1 cells;. HRCA system can be used to detect simulated natural water samples and natural water samples. (2) HRCA amplification technique and fluorescence quantitative analysis (quantitative real-time PCR,) were studied. RT-qPCR technique combined with the quantitative detection method of target algae was used to establish the fluorescence quantification-super-branching loop amplification (quantitative real-time hyper-branched rolling circle amplification,RT-qHRCA) and RT-qPCR detection system of Phaeodactylum tenuifolia, respectively. The specificity and sensitivity of the two amplification systems were tested, and the two amplification methods were evaluated by simulating the natural water samples. The results showed that the RT-qHRCA system could detect 1.4 cells, while the RT-qPCR system could only detect 7.5 cells, and the RT-qHRCA system could effectively detect the simulated natural water samples. The LSU rDNA was cloned and sequenced. According to the principle of PLP design, the PLP needed for HRCA amplification was designed manually, and the primers for loop mediated isothermal amplification (loop-mediated isothermal amplification,LAMP) were designed by Primerexplore. The detection system of HRCA and LAMP was established, the specificity and sensitivity of the two systems were tested after optimization, and the application of the two amplification methods was evaluated by simulating the natural water samples. The results showed that the specificity of the HRCA system was higher than that of the LAMP amplification system, and the sensitivity of the two amplification methods could reach 20 fg, cell detection limit of 10 cells;. And the two systems were not affected by non-target genomes. (4) the LSU rDNA was cloned and sequenced, and its target sequence was taken as the target. According to the design principle of PLP, the detection systems of HRCA and dHRCA for HRCA and double-cut hyperbranched (double nick hyper-branched rolling circle amplification,dHRCA were established, and the detection system was optimized. The method was evaluated by simulating the natural water samples. The results showed that both HRCA and dHRCA amplification system had high specificity, and the sensitivity of HRCA system was higher than that of dHRCA system.
【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：X55;X835
本文編號(hào)：2425198
[Abstract]:The outbreak of red tide consumes a great deal of human, material and financial resources every year in the world, especially the red tide caused by poisonous algae. Its destructive power is destructive to aquaculture and marine ecological environment, so it has a high specificity. It is necessary to detect red tide algae with high sensitivity. In this study, three toxic algae species of (Amphidinium carterae), (Heterosigma akashiwo), (Heterosigma akashiwo), were used as target algae, and hyper-branched rolling circle amplification, amplification (hyper-branched rolling circle amplification,) was studied. The application of HRCA technique in the detection of marine toxin producing algae. (1) the cloning and sequencing of rDNA sequence of (ribosomal large subunit,LSU, a large ribosomal subunit, was carried out on the basis of DNA sequencing. The result of sequencing was compared with the relative sequence to obtain the specific sequence. According to the design principle of locked probe (padlock probe,PLP), the specific PLP, was designed to establish the detection system of HRCA of Euglans roxburghii, and the detection system was optimized. The method was evaluated by simulating the natural water sample and the natural water sample. The results showed that the optimum joining temperature and amplification temperature of HRCA reaction were 59 擄C and 61 擄C, respectively, and the yield increased rapidly with the extension of time. The established HRCA system has high specificity and sensitivity. The sensitivity of plasmid detection is 1 cells;. The detection limit of the crude extract of 2 fg, cells is 1 cells;. HRCA system can be used to detect simulated natural water samples and natural water samples. (2) HRCA amplification technique and fluorescence quantitative analysis (quantitative real-time PCR,) were studied. RT-qPCR technique combined with the quantitative detection method of target algae was used to establish the fluorescence quantification-super-branching loop amplification (quantitative real-time hyper-branched rolling circle amplification,RT-qHRCA) and RT-qPCR detection system of Phaeodactylum tenuifolia, respectively. The specificity and sensitivity of the two amplification systems were tested, and the two amplification methods were evaluated by simulating the natural water samples. The results showed that the RT-qHRCA system could detect 1.4 cells, while the RT-qPCR system could only detect 7.5 cells, and the RT-qHRCA system could effectively detect the simulated natural water samples. The LSU rDNA was cloned and sequenced. According to the principle of PLP design, the PLP needed for HRCA amplification was designed manually, and the primers for loop mediated isothermal amplification (loop-mediated isothermal amplification,LAMP) were designed by Primerexplore. The detection system of HRCA and LAMP was established, the specificity and sensitivity of the two systems were tested after optimization, and the application of the two amplification methods was evaluated by simulating the natural water samples. The results showed that the specificity of the HRCA system was higher than that of the LAMP amplification system, and the sensitivity of the two amplification methods could reach 20 fg, cell detection limit of 10 cells;. And the two systems were not affected by non-target genomes. (4) the LSU rDNA was cloned and sequenced, and its target sequence was taken as the target. According to the design principle of PLP, the detection systems of HRCA and dHRCA for HRCA and double-cut hyperbranched (double nick hyper-branched rolling circle amplification,dHRCA were established, and the detection system was optimized. The method was evaluated by simulating the natural water samples. The results showed that both HRCA and dHRCA amplification system had high specificity, and the sensitivity of HRCA system was higher than that of dHRCA system.
【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：X55;X835
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