四种常见化脓性感染病原菌的分子检测
发布时间：2018-07-17 02:26
【摘要】：大肠杆菌、粪肠球菌、金黄色葡萄球菌和表皮葡萄球菌是常见的化脓性感染病原体,可以引起皮肤、皮下软组织、深部组织的化脓性感染乃至内脏器官的脓肿,也能引起脓毒血症。常对多种抗生素呈不同程度的耐受。在临床检验诊断中,快速准确的检测出病原微生物至关重要。现有的检测方法有分离培养法、免疫学检测法和分子生物学检测方法。目前在临床应用中分离培养法还是菌种检测的金标准,但其耗费时间长,操作繁琐,对检测人员要求较高；免疫学检测法麻较烦需要专门的技术和工具。故本研究从分子生物学角度探索大肠杆菌、粪肠球菌、金黄色葡萄球菌和表皮葡萄球菌的检测方法,建立了四种病原体的PCR检测体系以及表皮葡萄球菌的LAMP检测。本研究对大肠杆菌、粪肠球菌、金黄色葡萄球菌和表皮葡萄球菌进行生物信息学分析,筛选出其特异基因。大肠杆菌、粪肠球菌、金黄色葡萄球菌各筛选出基因分别为：LafF(GI:12887815)、TranR(GI:13347275)和 PurE(GI:12329975),表皮葡萄球菌筛选出两个基因为：LysE(GI:3240523)和DeoR(GI:3240916)。针对特异基因设计引物,在单重PCR扩增中,对Mg2+浓度和退火温度进行优化,得到每对引物最佳的Mg2+浓度和退火温度的组合,选择优化好的条件进行扩增,评价检测体系的特异性和灵敏度。利用单重PCR方法对大肠杆菌、粪肠球菌、金黄色葡萄球菌和表皮葡萄球菌各10株,肺炎克雷伯菌、弗劳地氏枸橼酸杆菌、伤寒杆菌、琼氏不动杆菌、溶血葡萄球菌和摩氏摩根菌各3株进行检测,结果显示只有大肠杆菌、粪肠球菌、金黄色葡萄球菌和表皮葡萄球菌出现了相应的预期大小的目的条带,其它菌株并未出现任何条带。这表明该检测体系具有非常好的特异性。利用单重PCR方法对大肠杆菌、粪肠球菌、金黄色葡萄球菌和表皮葡萄球菌分别进行梯度检测,结果显示大肠杆菌、粪肠球菌、金黄色葡萄球菌和表皮葡萄球菌检测下限可达到2个/反应。这表明该检测体系具有很高的灵敏度。进行多重PCR扩增时,在单重PCR条件优化的基础上,筛选合适的Mg2+浓度和退火温度组合,最终确定50μL反应体系的反应条件为：Taq酶(5U/μL) 0.3μL、dNTP混合物(2.5mM)4μL、10×PCR Buffer 5μL、MgCl2(25mM)3.2μL、四种菌的上、下游引物(10μM)各2μL,反应的退火温度为57℃,延伸时间为20秒。在上述反应条件下,评价此多重PCR检测体系的特异性和灵敏度。反应结果显示大肠杆菌、粪肠球菌、金黄色葡萄球菌和表皮葡萄球菌均能扩增出目的条带；大肠杆菌、粪肠球菌、金黄色葡萄球菌和表皮葡萄球菌多重PCR检测的下限为2个／反应。我们还应用LAMP等温扩增的方法对表皮葡萄球菌的DeoR(GI:3240916)基因进行鉴定,通过优化反应条件,建立了LAMP检测体系。最终确定25μL反应体系的反应条件为：Bst酶0.5μL、10×Thermpol 2.5μL、MgCl2(25mM) 4μL、dNTP混合物(2.5M) 4μL,内引物FIP和BIP各2μL、外引物F3和B3各0.5μL、甜菜碱(16mol/L)2.5μL,反应温度为65℃,反应时间为75分钟。反应结果显示只有表皮葡萄球菌出现扩增产物,其它菌种并未出现,表明该环介导等温扩增建测体系有较好的特异性；表皮葡萄球菌的检测下限为2个/反应,表明该环介导等温扩增建测体系有较高的灵敏度。总之,我们通过PCR和LAMP手段对常见化脓性感染病原体大肠杆菌、粪肠球菌、金黄色葡萄球菌和表皮葡萄球菌进行快速检测,检测用基因为首次应用于各自菌种鉴定,建立的检测方法简单、快速、灵敏度和特异性高。
[Abstract]:Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis are common pyogenic infection pathogens, which can cause skin, subcutaneous soft tissue, suppurative infection of deep tissue and abscess of visceral organs, and can also cause sepsis. It often tolerates a variety of antibiotics. In clinical diagnosis, fast The rapid and accurate detection of pathogenic microbes is very important. The existing methods include isolation and culture, immunological detection and molecular biological detection. In clinical application, the separation culture method or the gold standard of strain detection is currently used, but it takes a long time, the operation is tedious, the requirement of the inspectors is higher, and the immunological detection method is more common. This study explored the methods of detection of Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis from the molecular biology point of view, established the PCR detection system of four pathogens and the LAMP detection of Staphylococcus epidermidis. This study was on Escherichia coli, Enterococcus faecalis and Staphylococcus aureus. The specific genes were screened out by bioinformatics analysis and Staphylococcus epidermidis. Escherichia coli, Enterococcus faecalis and Staphylococcus aureus were selected as LafF (GI:12887815), TranR (GI:13347275) and PurE (GI:12329975). Staphylococcus epidermidis screened two bases because of LysE (GI:3240523) and DeoR (GI:3240916). In the single weight PCR amplification, the Mg2+ concentration and annealing temperature were optimized in the single weight amplification. The optimum combination of Mg2+ concentration and annealing temperature for each pair of primers was obtained. The optimized conditions were selected to amplify and evaluate the specificity and sensitivity of the detection system. Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus aureus were used by single weight PCR method. 10 strains of Staphylococcus epidermidis, Klebsiella pneumoniae, citrate citrate, Bacillus typhi, Acinetobacter jonmannii, Staphylococcus haemolyticus and Morgan bacteria were detected in 3 strains. The results showed that only Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis had the corresponding expected target bands, The other strains did not appear any bands. This showed that the detection system was very specific. The gradient detection of Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis by single weight PCR method was detected respectively. The results showed that the lower limits of Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis were detected. It can reach 2 / reaction. This shows that the detection system has high sensitivity. On the basis of the optimization of the single PCR condition, the suitable Mg2+ concentration and the annealing temperature combination are screened on the basis of the optimization of the single PCR condition. The reaction conditions of the reaction system are as follows: the Taq enzyme (5U/ mu L) 0.3 mu L, the dNTP mixture (2.5mM) 4 mu L, 10 * PCR 5 micron. L2 (25mM) 3.2 mu L, four kinds of bacteria, the downstream primers (10 mu M) each 2 mu L, the annealing temperature of the reaction was 57, 20 seconds. Under the above reaction conditions, the specificity and sensitivity of the multiple PCR detection system were evaluated. The results showed that the Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis could amplify the target The lower limit of multiple PCR detection of Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis was 2 / reaction. We also identified the DeoR (GI:3240916) gene of Staphylococcus epidermidis by LAMP isothermal amplification. By optimizing the reaction conditions, the LAMP detection system was established. Finally, the 25 L reaction system was determined. The reaction conditions were as follows: Bst enzyme 0.5 mu L, 10 x Thermpol 2.5 mu L, MgCl2 (25mM) 4 mu L, dNTP mixture (2.5M) 4 micron L, primers FIP and BIP each 2 mu, external primers and 0.5 microns each, 2.5 micron of betaine, reaction temperature of 65, and 75 minutes. The reaction results showed that only Staphylococcus epidermidis appeared amplification products and other strains did not produce It shows that the ring mediated isothermal amplification system has a good specificity. The detection limit of Staphylococcus epidermidis is 2 / reaction. It shows that the ring mediated isothermal amplification system has high sensitivity. In conclusion, we use PCR and LAMP to detect the pathogenic Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus faecalis. And the rapid detection of Staphylococcus epidermidis, the detection gene is first applied to the identification of different strains of bacteria. The detection method established is simple, rapid, sensitive and specific.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5
本文编号：2128605
[Abstract]:Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis are common pyogenic infection pathogens, which can cause skin, subcutaneous soft tissue, suppurative infection of deep tissue and abscess of visceral organs, and can also cause sepsis. It often tolerates a variety of antibiotics. In clinical diagnosis, fast The rapid and accurate detection of pathogenic microbes is very important. The existing methods include isolation and culture, immunological detection and molecular biological detection. In clinical application, the separation culture method or the gold standard of strain detection is currently used, but it takes a long time, the operation is tedious, the requirement of the inspectors is higher, and the immunological detection method is more common. This study explored the methods of detection of Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis from the molecular biology point of view, established the PCR detection system of four pathogens and the LAMP detection of Staphylococcus epidermidis. This study was on Escherichia coli, Enterococcus faecalis and Staphylococcus aureus. The specific genes were screened out by bioinformatics analysis and Staphylococcus epidermidis. Escherichia coli, Enterococcus faecalis and Staphylococcus aureus were selected as LafF (GI:12887815), TranR (GI:13347275) and PurE (GI:12329975). Staphylococcus epidermidis screened two bases because of LysE (GI:3240523) and DeoR (GI:3240916). In the single weight PCR amplification, the Mg2+ concentration and annealing temperature were optimized in the single weight amplification. The optimum combination of Mg2+ concentration and annealing temperature for each pair of primers was obtained. The optimized conditions were selected to amplify and evaluate the specificity and sensitivity of the detection system. Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus aureus were used by single weight PCR method. 10 strains of Staphylococcus epidermidis, Klebsiella pneumoniae, citrate citrate, Bacillus typhi, Acinetobacter jonmannii, Staphylococcus haemolyticus and Morgan bacteria were detected in 3 strains. The results showed that only Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis had the corresponding expected target bands, The other strains did not appear any bands. This showed that the detection system was very specific. The gradient detection of Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis by single weight PCR method was detected respectively. The results showed that the lower limits of Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis were detected. It can reach 2 / reaction. This shows that the detection system has high sensitivity. On the basis of the optimization of the single PCR condition, the suitable Mg2+ concentration and the annealing temperature combination are screened on the basis of the optimization of the single PCR condition. The reaction conditions of the reaction system are as follows: the Taq enzyme (5U/ mu L) 0.3 mu L, the dNTP mixture (2.5mM) 4 mu L, 10 * PCR 5 micron. L2 (25mM) 3.2 mu L, four kinds of bacteria, the downstream primers (10 mu M) each 2 mu L, the annealing temperature of the reaction was 57, 20 seconds. Under the above reaction conditions, the specificity and sensitivity of the multiple PCR detection system were evaluated. The results showed that the Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis could amplify the target The lower limit of multiple PCR detection of Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis was 2 / reaction. We also identified the DeoR (GI:3240916) gene of Staphylococcus epidermidis by LAMP isothermal amplification. By optimizing the reaction conditions, the LAMP detection system was established. Finally, the 25 L reaction system was determined. The reaction conditions were as follows: Bst enzyme 0.5 mu L, 10 x Thermpol 2.5 mu L, MgCl2 (25mM) 4 mu L, dNTP mixture (2.5M) 4 micron L, primers FIP and BIP each 2 mu, external primers and 0.5 microns each, 2.5 micron of betaine, reaction temperature of 65, and 75 minutes. The reaction results showed that only Staphylococcus epidermidis appeared amplification products and other strains did not produce It shows that the ring mediated isothermal amplification system has a good specificity. The detection limit of Staphylococcus epidermidis is 2 / reaction. It shows that the ring mediated isothermal amplification system has high sensitivity. In conclusion, we use PCR and LAMP to detect the pathogenic Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus faecalis. And the rapid detection of Staphylococcus epidermidis, the detection gene is first applied to the identification of different strains of bacteria. The detection method established is simple, rapid, sensitive and specific.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5
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