超分枝滚环扩增技术应用于几种海洋产毒微藻的检测
发布时间：2019-02-17 13:09
【摘要】：全世界每年因赤潮的爆发消耗了大量的人力、物力和财力,尤其是产毒藻种引发的赤潮,其破坏力对水产养殖业和海洋生态环境均是毁灭性的,因此建立高特异性、高灵敏度的赤潮藻检测方法非常必要。本研究以强壮前沟藻(Amphidinium carterae)、赤潮异弯藻(Heterosigma akashiwo)、米氏凯伦藻(Karenia mikimotoi)三种产毒藻种为目标藻,探讨了超分枝滚环扩增(hyper-branched rolling circle amplification,HRCA)技术在海洋产毒藻检测中的应用。(1)以强壮前沟藻为研究对象,通过核糖体大亚基(ribosomal large subunit,LSU)rDNA序列的克隆及测序,将测序结果与相关序列经比对分析获得种特异性序列,并根据锁式探针(padlock probe,PLP)的设计原则设计特异性的PLP,建立强壮前沟藻HRCA检测体系,对检测体系进行优化、特异性验证和灵敏度测试,并最终通过模拟自然水样和自然水样对建立的方法进行了评价。结果表明:HRCA反应的最佳连接温度和扩增温度分别是59°C和61°C,并且随着时间的延长产量迅速增加;建立的强壮前沟藻HRCA体系具有极高的特异性和灵敏度,质粒检测灵敏度为2 fg,细胞粗提液检测极限为1 cells;HRCA体系可以对模拟自然水样和自然水样进行有效检测。(2)以强壮前沟藻为目标藻,探讨了将HRCA扩增技术与荧光定量(quantitative real-time PCR,RT-qPCR)技术相结合用于目标藻的定量检测方法,分别建立了强壮前沟藻的荧光定量-超分枝滚环扩增(quantitative real-time hyper-branched rolling circle amplification,RT-qHRCA)和RT-qPCR检测体系,对两种扩增体系进行了特异性和灵敏度测试,并最终通过模拟自然水样对两种扩增方法进行了评价。结果表明:RT-qHRCA体系可以检测到1.4 cells,而RT-qPCR体系只能检测到7.5 cells,并且RT-qHRCA体系可以对模拟自然水样进行有效检测。(3)以赤潮异弯藻为目标藻,对其LSU rDNA进行克隆测序,并以其为靶序列,分别根据PLP设计原则手工设计HRCA扩增所需的PLP和采用在线设计软件Primerexplore设计环介导恒温扩增(loop-mediated isothermal amplification,LAMP)所需引物,建立了赤潮异弯藻的HRCA和LAMP检测体系,优化后对两种体系进行了特异性和灵敏度测试,并最终通过模拟自然水样对两种扩增方法进行了应用评价。结果表明:本实验建立的HRCA体系的特异性要高于LAMP扩增体系;两种扩增方法的质粒检测灵敏度可达20 fg,细胞检测极限可达10 cells;并且两种体系均不受非目标基因组的影响。(4)以米氏凯伦藻为目标藻,对其LSU rDNA进行克隆测序,并以其为靶序列,分别根据PLP设计原则设计HRCA和双切口超分枝滚环扩增(double nick hyper-branched rolling circle amplification,dHRCA)所需的PLP,建立了米氏凯伦藻HRCA和dHRCA检测体系,对检测体系进行了优化、特异性验证和灵敏度测试,并最终通过模拟自然水样对建立的方法进行了评价。结果表明:HRCA和dHRCA扩增体系均具有极高的特异性;HRCA体系灵敏度要高于dHRCA体系。
[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 掳C and 61 掳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.
【学位授予单位】：哈尔滨工业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：X55;X835
本文编号：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 掳C and 61 掳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.
【学位授予单位】：哈尔滨工业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：X55;X835
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