基因传感及免疫技术在农业废物堆肥化过程检测中的应用研究

发布时间：2018-06-09 15:59

本文选题：酶联免疫 + 毒莠定　；参考：《湖南大学》2015年博士论文

【摘要】：堆肥化在农业废物处理中具有较好的效果。农业废物里的农药残留不仅影响堆肥化进程,还会通过土壤回用造成二次污染。酶联免疫吸附法灵敏度高,操作简便快捷,应用于土壤和堆肥过程中痕量农药残留检测,有助于优化堆肥进程,控制堆肥产品的质量。农业废物堆肥中的关键内容和限速步骤是木质纤维素的高效降解,检测参与降解的活性酶及其编码基因对堆肥化进程有帮助。基因电化学传感技术在复杂体系中检测目标基因,具有特异性强和高灵敏度的优点,可以实现对堆肥体系中功能基因的痕量快速检测,不仅能够更好的揭示微生物对堆体环境的反馈,更能为从机制上了解和调整堆肥化中有关木质纤维素的降解提供更准确的信息。本文引入了竞争检测机制研究免疫技术和基因传感技术在农业废物堆肥过程中的应用,用酶联免疫吸附法实现对除草剂毒莠定的检测；同时制备了自组装基因分子膜修饰的金电极和丝网印刷电极,结合多种传统分子生物学方法,用于堆肥过程中木素过氧化物酶和漆酶功能基因的高灵敏特异性的电化学传感检测。1、建立了间接竞争酶联免疫吸附法检测土壤及堆肥样品中的毒莠定。通过固定包被抗原,优化酶标二抗和毒莠定多克隆抗体的工作浓度,利用包被抗原和待测溶液中的毒莠定互相竞争与抗体特异性结合,酶标二抗检出并放大信号,实现对稻田和农业废物堆肥体系的农药残留毒莠定的检测。优化了酶标二抗和抗体的最佳工作浓度均为1：500(V/V)。在保持酶标二抗和毒莠定抗体最佳工作浓度的待测溶液中,固相抗原毒莠定与待测溶液中毒莠定形成竞争关系,获得检测线性范围是0.07μg/mL～0.7μg/mL,检测下限为5ng/mL。该方法应用于检测土壤浸提液和堆肥腐熟样品浸提液中的毒莠定,样品基质对检测结果无显著干扰,回收率和变异系数均符合农药残留分析的要求,具有很高的灵敏度和重复性,可实现快捷方便的批量样品检测。2、通过烧灼、打磨等物理方法利用玻璃管和金丝自制了金电极,利用Au-S键把单链DNA固定在金电极表面,制得稳定有序的自组装基因分子膜,用于竞争式杂交电化学传感检测人工合成的黄孢原毛平革茵木素过氧化物酶的基因。通过差分脉冲伏安法、循环伏安法、交流阻抗法和电流-时间曲线法,优化了自组装时间和信号探针的最佳响应浓度,研究目标基因的线性检测范围和再生性能。结果表明：修饰金电极最优自组装时间为15 h,信号探针的最佳响应浓度为0.51 nmol/L,目标基因浓度范围为7.51×10-12-1.05×10-9 mol/L,检出下限为7.51x10-13 mol/L,可实现目标基因的低浓度检测。3、设计基因探针和PCR引物,利用基因提取、聚合酶链反应、琼脂糖凝胶电泳、基因纯化和核酸杂交等分子生物学方法,获得纯培养以及堆肥发酵体系中黄孢原毛平革菌(Phanerochaete chrysosporium)木素过氧化物酶天然DNA序列,长度为265 bp,用金电极自组装基因分子膜对它进行电化学杂交传感检测,结果与用紫外分光光度法的测定结果差别不大,平均变异系数和回收率分别为7.8%和100.97%。同时筛选了高特异性和信号放大能力强的HRP-SA作为酶标记物。该方法能实现对堆肥样品中黄孢原毛平格菌lip基因的快速灵敏检测,具有较高的特异性,精密度和准确性较好。4、在模拟农业废物堆肥的培养发酵体系接种凤尾侧耳(Pleurotus pulmonarius),在不同阶段测定漆酶酶活,同时提取基因组DNA,对比分析两者的动态变化发展趋势。同时制备了丝网印刷自组装基因分子膜,电化学表征其在外加电压下稳定性良好,用于杂交传感检测漆酶特异性编码人工合成DNA,线性检测范围为0.25～17 ng/mL,检测下限为9pg/mL。该方法应用于检测凤尾侧耳Lac基因PCR产物(339bp),结果与用紫外分光光度法的测定结果差别不大,精密度和准确性较好,并具有较高的特异性,为分析堆肥木质素降解酶的基因表达规律及微生物种群变化动态和机理提供参考。
[Abstract]:Composting has a good effect in the treatment of agricultural waste. Pesticide residues in agricultural waste not only affect the process of composting, but also cause two pollution through soil reuse. The enzyme linked immunosorbent assay is sensitive and easy to operate. It is applied to the detection of Trace Pesticide Residues in the process of soil and composting, which is helpful to optimize the process of composting and control the process of composting. The quality of the composting products. The key content and speed limiting step in the agricultural waste compost is the efficient degradation of the lignocellulose. The detection of the active enzymes involved in the degradation and the encoding genes are helpful to the process of composting. The rapid detection of functional genes in the composting system can not only reveal the feedback of microorganism to the environment of the heap, but also provide more accurate information for the mechanism of understanding and adjusting the degradation of lignocellulose in composting. In the process of agricultural waste composting, enzyme linked immunosorbent assay (ELISA) was used to detect herbicide and atrazine. At the same time, the gold electrode and screen printing electrode modified by self assembled gene molecular membrane were prepared, and combined with many traditional molecular biological methods, the high sensitivity of the lignin peroxidase and laccase function gene in the process of composting was used. .1 was detected by electrochemical sensing of the opposite sex, and the indirect competitive enzyme linked immunosorbent assay was established for the detection of atrazine in soil and composting samples. By immobilization of antigen, the working concentration of enzyme two and atrazine polyclonal antibody was optimized, and the binding of atrazine in the coated antigen and the atrazine in the solution was combined with the antibody specificity, and the enzyme label two Detection and amplification of signals to detect the pesticide residues in the rice field and the agricultural waste composting system. The optimum working concentration of the enzyme standard two anti and antibody is 1:500 (V/V). In the solution to keep the best working concentration of the enzyme standard two and the atrazine, the solid phase antigen and the atrazine are in the atrazine. In the competitive relationship, the linear range of detection is 0.07 g/mL to 0.7 mu g/mL, and the detection limit is 5ng/mL.. The method is applied to the detection of atrazine in the soil leaching solution and the leaching solution of composting samples. The sample matrix has no significant interference to the test results, and the recovery and coefficient of variation are both consistent with the requirements of pesticide residue analysis, and have high sensitivity. Degree and reproducibility can be used to detect.2 with quick and convenient mass sample. By means of burning, grinding and other physical methods, the gold electrode is made by glass tube and gold wire, and the single strand DNA is immobilized on the surface of gold electrode by Au-S key. The stable and orderly molecular membrane of self assembled gene is prepared, and it is used for the detection of synthetic yellow spores by competitive hybrid electrochemical sensing. The gene of the lignin peroxidase of the primary gram was optimized by differential pulse voltammetry, cyclic voltammetry, AC impedance method and current time curve method to optimize the self assembly time and the optimal response concentration of the signal probe. The linear detection range and regeneration performance of the target gene were studied. The results showed that the optimal self-assembly time of the modified gold electrode was obtained. For 15 h, the best response concentration of the signal probe is 0.51 nmol/L, the target gene concentration range is 7.51 x 10-12-1.05 x 10-9 mol/L, the detection limit is 7.51x10-13 mol/L, and the low concentration of the target gene can be detected for.3, the gene probe and PCR primers are designed, and the gene extraction, the agarose gel electrophoresis, the gene purification and the nucleic acid are used for the gene extraction, the agarose gel electrophoresis, the gene purification and the nucleic acid. The natural DNA sequence of the lignin peroxidase (Phanerochaete Chrysosporium) in the pure culture and compost fermentation system was obtained by hybridization and other molecular biological methods. The length of the DNA sequence was 265 BP, and was detected by electrochemical hybridization with the gold electrode self assembled gene molecular membrane. The difference was not significant. The average coefficient of variation and recovery were 7.8% and 100.97%., respectively, and the high specificity and strong signal amplification ability of HRP-SA were selected as enzyme markers. This method can detect the lip gene of P. xanospora in the compost samples quickly and sensitively. It has high specificity, precision and accuracy is better.4, in the model. The cultivation and fermentation system for agricultural waste composting was inoculated with Pleurotus pulmonarius, laccase activity was measured at different stages and genomic DNA was extracted, and the dynamic change trend of the two was compared and analyzed. At the same time, the self assembled molecular membrane of silk screen printing was prepared, and the electrochemical characterization of the membrane was stable under the applied voltage. Hybrid sensing detection laccase specific encoding of DNA, linear detection range of 0.25 to 17 ng/mL, the detection limit is 9pg/mL., the method is applied to the detection of Lac gene PCR product (339bp) of the side ear of the phoenix tail. The result is not very different from the UV spectrophotometric method, the precision and accuracy are better, and it has high specificity. It can provide reference for gene expression regulation of lignin degrading enzymes and microbial population dynamics and mechanism.
【学位授予单位】：湖南大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S141.4;X71

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