空气中3种常见病原菌快速定量检测方法研究

发布时间：2018-01-16 21:46

本文关键词：空气中3种常见病原菌快速定量检测方法研究　出处：《河北大学》2012年硕士论文　论文类型：学位论文

【摘要】：微生物主要以气溶胶的形式存在于空气中，气溶胶颗粒较小，容易造成空气污染。空气微生物不仅是室内空气的污染源，而且是影响室内工作人员健康的一个重要因素。本论文通过免培养方法研究了实验室空气细菌的组成和多样性，在此基础上建立了大肠杆菌、金黄色葡萄球菌、鲍曼氏不动杆菌这三种空气常见病原微生物的快速定量检测方法。采用微孔滤膜法采集实验室空气环境中的微生物，，根据滤膜孔径、采样时间等因素设计空气样品采集方案，发现适宜的滤膜孔径为0.22μm，采样时间和气流速度对微生物的采集效率的影响不明显，但需根据气体流速选择适当的采样时间，以保证得到足够数量用于DNA提取的微生物；通过掺菌法比较3种DNA提取试剂盒和DNA快速提取液的提取效果，发现土壤基因组DNA快速提取试剂盒的提取效果较好，但对金黄色葡萄球菌DNA提取效率较低，加入溶葡球菌酶和改进操作后可满足空气样品宏基因组DNA提取；采用16S rDNA克隆文库法分析了空气样品细菌的多样性组成：包括芽孢杆菌属(Bacillus)(60.3%)、葡萄球菌属(Staphylococcus)(1.2%)、不动杆菌属(Acinetobacter)(19.4%)、副球菌属(Paracoccus)(14.9%)、肠杆菌属(Enterobacter)(0.7%)、棒状杆菌属(Corynebacterium)(0.7%)、贪铜菌属(Cupriavidus)(0.5%)、赛托氏菌属(Schlegelella)(0.5%)、鞘氨醇盒菌属(Sphingopyxis)(0.5%)、黄单胞菌属(Xanthomonadaceae)(0.5%)、微枝形杆菌属(Microvirga)(0.2%)、鞘氨醇单胞菌属(Sphingomonas)(0.2%)、非培养的假单胞菌目(uncultured Pseudomonadales)(0.2%)和非培养的红细菌目(unculturedRhodobacterales)(0.2%)。根据多样性分析结果，选择大肠杆菌、金黄色葡萄球菌和鲍曼氏不动杆菌为空气中待测病原菌，大量调取这三种菌的不同基因的序列，利用BioEdit、primer premier5.0、DNAstar、DNAuser、Annbyb222等软件设计和筛选出这三种菌的特异性强、覆盖度高的引物用于荧光定量PCR检测，并用筛选得到的特异引物制作了三种空气常见病原菌的荧光定量PCR程序和定量曲线，建立了这三种空气病原菌的荧光定量PCR检测方法。
[Abstract]:Microbes mainly exist in the air in the form of aerosols, aerosol particles are small, which is easy to cause air pollution, air microorganisms are not only the indoor air pollution sources. In this paper, the composition and diversity of bacteria in the air of laboratory were studied by the method of non-culture, and the Escherichia coli and Staphylococcus aureus were established on this basis. Rapid quantitative detection of Acinetobacter baumannii, three common airborne pathogens. The microporous membrane method was used to collect microorganism in laboratory air environment. According to the factors such as membrane pore diameter and sampling time, the air sample sampling scheme was designed. The suitable filter membrane aperture was found to be 0.22 渭 m. The effect of sampling time and airflow velocity on the collection efficiency of microorganisms is not obvious, but the appropriate sampling time should be selected according to the gas flow rate to ensure that a sufficient number of microbes used for DNA extraction can be obtained. The results of three kinds of DNA extraction kit and DNA rapid extraction solution were compared by the method of mixed bacteria. It was found that the extraction effect of soil genomic DNA rapid extraction kit was better than that of soil genomic DNA rapid extraction kit. However, the extraction efficiency of DNA from Staphylococcus aureus was relatively low. The extraction of macro genomic DNA from air samples could be satisfied with the addition of lysostaphylococcase and the improved operation. The diversity of bacteria in air samples was analyzed by 16s rDNA clone library, including Bacillus sp. 60.3). Staphylococcus spp 1.2 and Acinetobacter 19. 4). Paracoccusis 14.9%, Enterobacter 0.7). Corynebacterium corynebacterium is 0. 7 and Cupriavidus is 0. 5). Sphingopyxischus (Sphingopyxischus). Xanthomonas sp., Xanthomonas adaceaeae 0.5 and Microvirgaa 0.2. Sphingomonas sphingomonas (Sphingomonas sphingomonas). The uncultured Pseudomonas sp. 0.2and the uncultured Rhodoptera (Orchidae). Unculturated Rhodobacter alesus 0.2k.According to the results of diversity analysis. Escherichia coli, Staphylococcus aureus and Acinetobacter baumannii were selected as pathogens to be tested in the air. The different genes of these three strains were sequenced by BioEdit. The primer premier5.0% DNA starDNA user#en0# Annbyb222 software was designed and screened out with strong specificity. The primers with high coverage were used to detect the fluorescence quantitative PCR, and the fluorescent quantitative PCR program and quantitative curve of three common airborne pathogens were made by using the selected specific primers. A fluorescence quantitative PCR method for the detection of these three airborne pathogens was established.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R122

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