本文選題：酶聯(lián)免疫 + 毒莠定��； 參考：《湖南大學(xué)》2015年博士論文

【摘要】：堆肥化在農(nóng)業(yè)廢物處理中具有較好的效果。農(nóng)業(yè)廢物里的農(nóng)藥殘留不僅影響堆肥化進(jìn)程,還會通過土壤回用造成二次污染。酶聯(lián)免疫吸附法靈敏度高,操作簡便快捷,應(yīng)用于土壤和堆肥過程中痕量農(nóng)藥殘留檢測,有助于優(yōu)化堆肥進(jìn)程,控制堆肥產(chǎn)品的質(zhì)量。農(nóng)業(yè)廢物堆肥中的關(guān)鍵內(nèi)容和限速步驟是木質(zhì)纖維素的高效降解,檢測參與降解的活性酶及其編碼基因?qū)Χ逊驶M(jìn)程有幫助�；螂娀瘜W(xué)傳感技術(shù)在復(fù)雜體系中檢測目標(biāo)基因,具有特異性強(qiáng)和高靈敏度的優(yōu)點(diǎn),可以實(shí)現(xiàn)對堆肥體系中功能基因的痕量快速檢測,不僅能夠更好的揭示微生物對堆體環(huán)境的反饋,更能為從機(jī)制上了解和調(diào)整堆肥化中有關(guān)木質(zhì)纖維素的降解提供更準(zhǔn)確的信息。本文引入了競爭檢測機(jī)制研究免疫技術(shù)和基因傳感技術(shù)在農(nóng)業(yè)廢物堆肥過程中的應(yīng)用,用酶聯(lián)免疫吸附法實(shí)現(xiàn)對除草劑毒莠定的檢測；同時制備了自組裝基因分子膜修飾的金電極和絲網(wǎng)印刷電極,結(jié)合多種傳統(tǒng)分子生物學(xué)方法,用于堆肥過程中木素過氧化物酶和漆酶功能基因的高靈敏特異性的電化學(xué)傳感檢測。1、建立了間接競爭酶聯(lián)免疫吸附法檢測土壤及堆肥樣品中的毒莠定。通過固定包被抗原,優(yōu)化酶標(biāo)二抗和毒莠定多克隆抗體的工作濃度,利用包被抗原和待測溶液中的毒莠定互相競爭與抗體特異性結(jié)合,酶標(biāo)二抗檢出并放大信號,實(shí)現(xiàn)對稻田和農(nóng)業(yè)廢物堆肥體系的農(nóng)藥殘留毒莠定的檢測。優(yōu)化了酶標(biāo)二抗和抗體的最佳工作濃度均為1：500(V/V)。在保持酶標(biāo)二抗和毒莠定抗體最佳工作濃度的待測溶液中,固相抗原毒莠定與待測溶液中毒莠定形成競爭關(guān)系,獲得檢測線性范圍是0.07μg/mL～0.7μg/mL,檢測下限為5ng/mL。該方法應(yīng)用于檢測土壤浸提液和堆肥腐熟樣品浸提液中的毒莠定,樣品基質(zhì)對檢測結(jié)果無顯著干擾,回收率和變異系數(shù)均符合農(nóng)藥殘留分析的要求,具有很高的靈敏度和重復(fù)性,可實(shí)現(xiàn)快捷方便的批量樣品檢測。2、通過燒灼、打磨等物理方法利用玻璃管和金絲自制了金電極,利用Au-S鍵把單鏈DNA固定在金電極表面,制得穩(wěn)定有序的自組裝基因分子膜,用于競爭式雜交電化學(xué)傳感檢測人工合成的黃孢原毛平革茵木素過氧化物酶的基因。通過差分脈沖伏安法、循環(huán)伏安法、交流阻抗法和電流-時間曲線法,優(yōu)化了自組裝時間和信號探針的最佳響應(yīng)濃度,研究目標(biāo)基因的線性檢測范圍和再生性能。結(jié)果表明：修飾金電極最優(yōu)自組裝時間為15 h,信號探針的最佳響應(yīng)濃度為0.51 nmol/L,目標(biāo)基因濃度范圍為7.51×10-12-1.05×10-9 mol/L,檢出下限為7.51x10-13 mol/L,可實(shí)現(xiàn)目標(biāo)基因的低濃度檢測。3、設(shè)計(jì)基因探針和PCR引物,利用基因提取、聚合酶鏈反應(yīng)、瓊脂糖凝膠電泳、基因純化和核酸雜交等分子生物學(xué)方法,獲得純培養(yǎng)以及堆肥發(fā)酵體系中黃孢原毛平革菌(Phanerochaete chrysosporium)木素過氧化物酶天然DNA序列,長度為265 bp,用金電極自組裝基因分子膜對它進(jìn)行電化學(xué)雜交傳感檢測,結(jié)果與用紫外分光光度法的測定結(jié)果差別不大,平均變異系數(shù)和回收率分別為7.8%和100.97%。同時篩選了高特異性和信號放大能力強(qiáng)的HRP-SA作為酶標(biāo)記物。該方法能實(shí)現(xiàn)對堆肥樣品中黃孢原毛平格菌lip基因的快速靈敏檢測,具有較高的特異性,精密度和準(zhǔn)確性較好。4、在模擬農(nóng)業(yè)廢物堆肥的培養(yǎng)發(fā)酵體系接種鳳尾側(cè)耳(Pleurotus pulmonarius),在不同階段測定漆酶酶活,同時提取基因組DNA,對比分析兩者的動態(tài)變化發(fā)展趨勢。同時制備了絲網(wǎng)印刷自組裝基因分子膜,電化學(xué)表征其在外加電壓下穩(wěn)定性良好,用于雜交傳感檢測漆酶特異性編碼人工合成DNA,線性檢測范圍為0.25～17 ng/mL,檢測下限為9pg/mL。該方法應(yīng)用于檢測鳳尾側(cè)耳Lac基因PCR產(chǎn)物(339bp),結(jié)果與用紫外分光光度法的測定結(jié)果差別不大,精密度和準(zhǔn)確性較好,并具有較高的特異性,為分析堆肥木質(zhì)素降解酶的基因表達(dá)規(guī)律及微生物種群變化動態(tài)和機(jī)理提供參考。
[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.
【學(xué)位授予單位】：湖南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：S141.4;X71

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本文編號：2000309