本文關(guān)鍵詞：空氣中3種常見病原菌快速定量檢測方法研究　出處：《河北大學》2012年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 16S rDNA克隆文庫 空氣細菌 組成和多樣性 病原菌 定量檢測

【摘要】：微生物主要以氣溶膠的形式存在于空氣中，氣溶膠顆粒較小，容易造成空氣污染。空氣微生物不僅是室內(nèi)空氣的污染源，而且是影響室內(nèi)工作人員健康的一個重要因素。本論文通過免培養(yǎng)方法研究了實驗室空氣細菌的組成和多樣性，在此基礎(chǔ)上建立了大腸桿菌、金黃色葡萄球菌、鮑曼氏不動桿菌這三種空氣常見病原微生物的快速定量檢測方法。 采用微孔濾膜法采集實驗室空氣環(huán)境中的微生物，，根據(jù)濾膜孔徑、采樣時間等因素設(shè)計空氣樣品采集方案，發(fā)現(xiàn)適宜的濾膜孔徑為0.22μm，采樣時間和氣流速度對微生物的采集效率的影響不明顯，但需根據(jù)氣體流速選擇適當?shù)牟蓸訒r間，以保證得到足夠數(shù)量用于DNA提取的微生物；通過摻菌法比較3種DNA提取試劑盒和DNA快速提取液的提取效果，發(fā)現(xiàn)土壤基因組DNA快速提取試劑盒的提取效果較好，但對金黃色葡萄球菌DNA提取效率較低，加入溶葡球菌酶和改進操作后可滿足空氣樣品宏基因組DNA提�。徊捎�16S rDNA克隆文庫法分析了空氣樣品細菌的多樣性組成：包括芽孢桿菌屬(Bacillus)(60.3%)、葡萄球菌屬(Staphylococcus)(1.2%)、不動桿菌屬(Acinetobacter)(19.4%)、副球菌屬(Paracoccus)(14.9%)、腸桿菌屬(Enterobacter)(0.7%)、棒狀桿菌屬(Corynebacterium)(0.7%)、貪銅菌屬(Cupriavidus)(0.5%)、賽托氏菌屬(Schlegelella)(0.5%)、鞘氨醇盒菌屬(Sphingopyxis)(0.5%)、黃單胞菌屬(Xanthomonadaceae)(0.5%)、微枝形桿菌屬(Microvirga)(0.2%)、鞘氨醇單胞菌屬(Sphingomonas)(0.2%)、非培養(yǎng)的假單胞菌目(uncultured Pseudomonadales)(0.2%)和非培養(yǎng)的紅細菌目(unculturedRhodobacterales)(0.2%)。根據(jù)多樣性分析結(jié)果，選擇大腸桿菌、金黃色葡萄球菌和鮑曼氏不動桿菌為空氣中待測病原菌，大量調(diào)取這三種菌的不同基因的序列，利用BioEdit、primer premier5.0、DNAstar、DNAuser、Annbyb222等軟件設(shè)計和篩選出這三種菌的特異性強、覆蓋度高的引物用于熒光定量PCR檢測，并用篩選得到的特異引物制作了三種空氣常見病原菌的熒光定量PCR程序和定量曲線，建立了這三種空氣病原菌的熒光定量PCR檢測方法。
[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.
【學位授予單位】：河北大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R122

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本文編號：1434961