摘要
随着环境污染问题的日益严重,环境中的病原微生物在人们的生活中到处可见,因对其严重性以及危害性的忽视,所以经常出现由致病菌引起的传染性疾病的爆发事件。传统的微生物学检验方法已不能满足这种突发性与规模性疾病检测的需要,所以,亟需开发出准确、快速、高效的环境中病原微生物的检测技术。此外,城镇污水处理厂污泥产量的剧增与普遍未得到有效处理处置的现状,同样成为十分严峻的环境问题。污泥中含有大量的病原微生物、重金属以及持久性有机物等有毒有害物质,可能会对地下水、土壤等造成二次污染,严重威胁到环境安全与人们的健康。所以,污泥有效的处理和处置问题,亦成为了众多环保人士关注的焦点。
本课题组在荧光分析检测方面有着扎实的研究基础,同时对卟啉及卟啉衍生物的合成与应用等方面同样开展了大量的基础性研究,并取得了一定的研究成果。基于上述两大环境问题以及本课题组的研究基础,本论文对环境中病原微生物的快速检测方法以及对剩余污泥的减量化方面进行探讨,研究的具体内容主要有以下五部分：
(1)建立了一种基于双探针串联DNA杂交以及时间分辨荧光分析法的大肠杆菌的快速检测方法。实验结果显示,固定在玻片表面的最佳捕获探针浓度为1.0×10-7mol/L,最适的杂交时间是10h,最适的杂交温度在43℃,最佳的洗涤时间为6min；该检测方法具有良好的特异性及灵敏度,且该方法还成功地运用到了土壤以及景观水中的大肠杆菌的检测中。此外,实验中检测的目标DNA是纯培养后从大肠杆菌菌体中提取的核酸成分,未经PCR的扩增以及DNA纯化过程,该方法能够达到令人满意的特异性和灵敏度,说明该方法具有操作方便、简单可行以及成本节约等优点,为其在环境微生物检测领域的进一步推广提供了有利的条件。
(2)在上述双探针串联DNA杂交模型的基础上,建立了一种对金黄色葡萄球菌的快速检测方法。优化了杂交温度、杂交时间以及洗涤时间等条件,实验结果显示,最适的杂交时间是9h,最适的杂交温度在53℃,最佳的洗涤时间为4.5min；该检测方法具有良好的特异性及灵敏度,且该方法还成功地检测了模拟污染的果汁与牛奶中的金黄色葡萄球菌的检测。
(3)采用微乳液法有效地合成了三种核壳型纳米颗粒,其中用IgG修饰铕配合物纳米颗粒具有良好的稳定性能。将其标记到DNA探针,与大肠杆菌及金黄色葡萄球菌的目标DNA杂交反应,结果表明,杂交体系的荧光信号强度提高不明显,可能由于纳米颗粒之间的团聚现象使得实验的重现性欠佳,若改善该纳米颗粒在水溶液中的分散性能,将会成为分子生物学中具发展前景的生物标记物。
(4)采用萘酰亚胺类物质EEN(N-羟乙基-4-(2-氨基乙烷氨基)-1,8萘酰亚胺)作为供体染料、花青素类物质Cy5作为受体染料,建立了一种基于荧光共振能量转移的核酸竞争杂交检测方法。考察了不同浓度的Acceptor probe-1与连接了EEN的Donor probe的杂交反应,得到令人满意的荧光能量转移的效果；在核酸竞争杂交的实验中,不同浓度的Target probe可以将与相对应浓度的Acceptor probe-1有效地杂交竞争替换,检测得到EEN荧光强度得以恢复以及Cy5荧光强度的减弱现象,建立了这种基于荧光共振能量转移的核酸竞争杂交检测方法。
(5)将合成的具有仿生细胞色素P450酶的金属铁卟啉Fe(TAPP)Cl应用于剩余污泥的厌氧消解处理过程,结果发现将铁卟啉可固定于普通玻璃珠表面,对于污泥的消解效果明显。铁卟啉的乙醇溶液作为仿生细胞色素P450单加氧酶在污泥消解过程中,最佳处理条件为：处理时间7h,处理温度在40℃,投量百分比约为1%。实验的操作条件较温和,对处理设备的要求简单经济,降低了其应用成本；同时,作为化学合成的仿生酶,对比天然酶而言在适用条件及范围上更具优势。
With the growing problem of environmental pollution, pathogenic microorganisms are present in our lives everywhere. Because of neglected the seriousness and danger of pathogens, there are many infectious disease outbreak events all around the world. The traditional microbiological testing methods can not meet the needs of rapid and efficient testing of the large scale and paroxysmal disease. Therefore, it is an urgent need to develop an accurate, fast and efficient method for detecting pathogenic microorganisms in environment. In addition, it is the other serious and easy to ignore environmental problem that the sludge production from urban sewage treatment plant are increasing but not to be sufficient treatment and disposal. As we all know, sludge contains large amounts of pathogenic microorganisms, heavy metals, persistent organic compounds and other toxic substances, which will cause secondary pollution and pollute the groundwater, soil and atmosphere. And there will be a serious threat to human health and environmental safety. For this reason, many environmentalists have started to focus on the sludge issue of effective treatment and disposal.
In our research group, we have an abundant foundation of fluorescence detection and also in the field of porphyrins and its derivatives have carry out a large number of basic research and applications, and made some research. And based on the above two major environmental issues and our advantage of research, this work will focus on rapid detection methods of pathogens and the reduction technology of excess sludge. The details are summarized as follows:
(1) A two-probe tandem DNA hybridization assay based on time-resolved fluorescence analysis method was employed to rapid detect Escherichia coli strain. The results showed that the optimal concentration of capture probe immobilized on glass surface was1.0×10-7mol/L, and the optimal hybridization time and temperature were10h and43℃, respectively. The detection method has good specificity and sensitivity, and the method also successfully applied to the soil and the landscape in the detection of E. coli. Furthermore, without PCR amplification and DNA purification process, the extracted nucleic acid was directly used to the hybridization process, and a satisfactory specificity and sensitivity result was obtained, which shows that the method is easy to operate, simple and feasible, and economical. It can be concluded that this method is a promising technique for monitoring the microorganisms.
(2) Based on the above method, it was re-established a fast and efficient way to detect Staphylococcus aureus. In this study, the hybridization temperature, hybridization time, washing time and other conditions were re-optimized. Results showed that the optimal hybridization time is9h, the optimal hybridization temperature at53℃,the best time for washing4.5min. The detection method has good specificity and sensitivity, and it also successfully detected a simulated contaminated fruit juice and milk in the detection of Staphylococcus aureus.
(3) Three kinds of core-shell nanoparticles were successfully synthesized by the microemulsion method. Among these nanoparticles, the one of europium complex Eu(TTA)3(5-NH2-phen) with IgG-modified has satisfactory fluorescence performance and package stability. The nanoparticle was labeled with DNA probes and employed to the hybridization reaction of target nucleic acid from the Escherichia coli and Staphylococcus aureus, respectively. Results showed that the fluorescence intensity of the two hybrid systems were slightly increased, but the reproducible performance of this detection systems were not better than that of the molecular europium complex. It may be due to agglomeration between nanoparticles. So, if improving the dispersion property of the nanoparticles in aqueous, it will be a very promising biomarker in the field of molecular biology.
(4) A fluorescence resonance energy transfer based competitive hybridization of nucleic acid detection methods was presented, in which naphthalimide EEN (N-hydroxyethyl4-(2-amino ethane) amino-1,8-naphth-alimide) was the donor dye and anthocyanin dye Cy5was the receptor substances. A series of Acceptor probe-1concentrations were employed to hybrid with Donor probe connected the EEN dyes. A satisfactory result of fluorescence resonance energy transfer was obtained. In the nucleic acid competition hybridization procedure, the Acceptor probe-1was effectively replaced by Target probe, which was appeared that the fluorescence intensity of EEN was restored and that of Cy5was weaken. In conclusion, it has successfully established fluorescence resonance energy transfer-based competitive hybridization of nucleic acid detection methods.
(5) The metal iron porphyrin Fe(TAPP)Cl synthesized by our team, as a biomimetic enzyme cytochrome P450, was applied to the excess sludge anaerobic digestion process. Results showed that the iron porphyrin can be immobilized on the surface of ordinary glass beads, and they worked effectively on the sludge digestion. When used the ethanol solution of iron porphyrin as biomimetic cytochrome monooxygenase in the sludge digestion process, the optimum digestion time was7h, the optimal treated temperature at40℃and the best percentage of dosage is about1%. It is apparent that this method has the advantages of easy operation, mild reaction condition and economical equipment. Therefore, compared with the natural enzymes, the chemical synthesis of biomimetic enzyme has the superiority in the operation condition and application scope.
引文
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