四種常見化膿性感染病原菌的分子檢測
發(fā)布時(shí)間：2018-07-17 02:26
【摘要】：大腸桿菌、糞腸球菌、金黃色葡萄球菌和表皮葡萄球菌是常見的化膿性感染病原體,可以引起皮膚、皮下軟組織、深部組織的化膿性感染乃至內(nèi)臟器官的膿腫,也能引起膿毒血癥。常對(duì)多種抗生素呈不同程度的耐受。在臨床檢驗(yàn)診斷中,快速準(zhǔn)確的檢測出病原微生物至關(guān)重要�，F(xiàn)有的檢測方法有分離培養(yǎng)法、免疫學(xué)檢測法和分子生物學(xué)檢測方法。目前在臨床應(yīng)用中分離培養(yǎng)法還是菌種檢測的金標(biāo)準(zhǔn),但其耗費(fèi)時(shí)間長,操作繁瑣,對(duì)檢測人員要求較高；免疫學(xué)檢測法麻較煩需要專門的技術(shù)和工具。故本研究從分子生物學(xué)角度探索大腸桿菌、糞腸球菌、金黃色葡萄球菌和表皮葡萄球菌的檢測方法,建立了四種病原體的PCR檢測體系以及表皮葡萄球菌的LAMP檢測。本研究對(duì)大腸桿菌、糞腸球菌、金黃色葡萄球菌和表皮葡萄球菌進(jìn)行生物信息學(xué)分析,篩選出其特異基因。大腸桿菌、糞腸球菌、金黃色葡萄球菌各篩選出基因分別為：LafF(GI:12887815)、TranR(GI:13347275)和 PurE(GI:12329975),表皮葡萄球菌篩選出兩個(gè)基因?yàn)椋篖ysE(GI:3240523)和DeoR(GI:3240916)。針對(duì)特異基因設(shè)計(jì)引物,在單重PCR擴(kuò)增中,對(duì)Mg2+濃度和退火溫度進(jìn)行優(yōu)化,得到每對(duì)引物最佳的Mg2+濃度和退火溫度的組合,選擇優(yōu)化好的條件進(jìn)行擴(kuò)增,評(píng)價(jià)檢測體系的特異性和靈敏度。利用單重PCR方法對(duì)大腸桿菌、糞腸球菌、金黃色葡萄球菌和表皮葡萄球菌各10株,肺炎克雷伯菌、弗勞地氏枸櫞酸桿菌、傷寒桿菌、瓊氏不動(dòng)桿菌、溶血葡萄球菌和摩氏摩根菌各3株進(jìn)行檢測,結(jié)果顯示只有大腸桿菌、糞腸球菌、金黃色葡萄球菌和表皮葡萄球菌出現(xiàn)了相應(yīng)的預(yù)期大小的目的條帶,其它菌株并未出現(xiàn)任何條帶。這表明該檢測體系具有非常好的特異性。利用單重PCR方法對(duì)大腸桿菌、糞腸球菌、金黃色葡萄球菌和表皮葡萄球菌分別進(jìn)行梯度檢測,結(jié)果顯示大腸桿菌、糞腸球菌、金黃色葡萄球菌和表皮葡萄球菌檢測下限可達(dá)到2個(gè)/反應(yīng)。這表明該檢測體系具有很高的靈敏度。進(jìn)行多重PCR擴(kuò)增時(shí),在單重PCR條件優(yōu)化的基礎(chǔ)上,篩選合適的Mg2+濃度和退火溫度組合,最終確定50μL反應(yīng)體系的反應(yīng)條件為：Taq酶(5U/μL) 0.3μL、dNTP混合物(2.5mM)4μL、10×PCR Buffer 5μL、MgCl2(25mM)3.2μL、四種菌的上、下游引物(10μM)各2μL,反應(yīng)的退火溫度為57℃,延伸時(shí)間為20秒。在上述反應(yīng)條件下,評(píng)價(jià)此多重PCR檢測體系的特異性和靈敏度。反應(yīng)結(jié)果顯示大腸桿菌、糞腸球菌、金黃色葡萄球菌和表皮葡萄球菌均能擴(kuò)增出目的條帶；大腸桿菌、糞腸球菌、金黃色葡萄球菌和表皮葡萄球菌多重PCR檢測的下限為2個(gè)／反應(yīng)。我們還應(yīng)用LAMP等溫?cái)U(kuò)增的方法對(duì)表皮葡萄球菌的DeoR(GI:3240916)基因進(jìn)行鑒定,通過優(yōu)化反應(yīng)條件,建立了LAMP檢測體系。最終確定25μL反應(yīng)體系的反應(yīng)條件為：Bst酶0.5μL、10×Thermpol 2.5μL、MgCl2(25mM) 4μL、dNTP混合物(2.5M) 4μL,內(nèi)引物FIP和BIP各2μL、外引物F3和B3各0.5μL、甜菜堿(16mol/L)2.5μL,反應(yīng)溫度為65℃,反應(yīng)時(shí)間為75分鐘。反應(yīng)結(jié)果顯示只有表皮葡萄球菌出現(xiàn)擴(kuò)增產(chǎn)物,其它菌種并未出現(xiàn),表明該環(huán)介導(dǎo)等溫?cái)U(kuò)增建測體系有較好的特異性；表皮葡萄球菌的檢測下限為2個(gè)/反應(yīng),表明該環(huán)介導(dǎo)等溫?cái)U(kuò)增建測體系有較高的靈敏度�？傊�,我們通過PCR和LAMP手段對(duì)常見化膿性感染病原體大腸桿菌、糞腸球菌、金黃色葡萄球菌和表皮葡萄球菌進(jìn)行快速檢測,檢測用基因?yàn)槭状螒?yīng)用于各自菌種鑒定,建立的檢測方法簡單、快速、靈敏度和特異性高。
[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.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R446.5
本文編號(hào)：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.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R446.5
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