利用寡核苷酸芯片檢測(cè)耐藥基因ampC的初步研究
發(fā)布時(shí)間：2018-08-16 13:13
【摘要】：抗生素的使用不當(dāng)導(dǎo)致細(xì)菌耐藥性的日益嚴(yán)重是臨床抗感染治療面臨的世界性難題。解決這一問(wèn)題的方法之一是根據(jù)感染細(xì)菌的耐藥性有針對(duì)性地使用抗生素。目前對(duì)細(xì)菌耐藥性的檢測(cè)仍然依賴傳統(tǒng)的藥敏實(shí)驗(yàn),其最大的缺點(diǎn)是診斷周期長(zhǎng),臨床醫(yī)生無(wú)法在感染早期對(duì)患者合理用藥。通過(guò)基因芯片技術(shù)直接檢測(cè)細(xì)菌耐藥基因,可以快速、敏感地確定細(xì)菌的耐藥性,為合理使用抗生素提供依據(jù)。革蘭陰性桿菌是目前臨床常見(jiàn)的致病菌,其治療主要依賴β-內(nèi)酰胺類抗生素。革蘭陰性桿菌耐受β-內(nèi)酰胺類抗生素主要由細(xì)菌產(chǎn)生的β-內(nèi)酰胺酶引起。β-內(nèi)酰胺酶種類繁多,其中較為重要的包括AmpC 酶、ESBLs 等。AmpC 酶屬于Bush 分類中的I 類酶或Ambler C 類酶,其編碼基因是ampC。我們發(fā)現(xiàn),不同種屬的細(xì)菌的ampC 基因序列顯著不同。 目的分析臨床革蘭陰性桿菌中ampC 耐藥結(jié)構(gòu)基因的分布與耐藥狀況的關(guān)系;在此基礎(chǔ)上建立起利用多重PCR 及寡核苷酸芯片技術(shù)檢測(cè)ampC 耐藥結(jié)構(gòu)基因的方法。 方法 1、標(biāo)本菌株收集來(lái)源于第三軍醫(yī)大學(xué)第一附屬醫(yī)院及兒童醫(yī)院檢驗(yàn)科2003 年10月至2004 年5 月臨床分離的菌株。 2、用PCR 檢測(cè)389 株革蘭陰性桿菌中ampC 基因的分布。 3、應(yīng)用頭孢西丁三維實(shí)驗(yàn)的方法檢測(cè)細(xì)菌產(chǎn)AmpC 酶的狀況。 4、應(yīng)用microscan walkaway-4.0 鑒定細(xì)菌的耐藥性。 5、建立多重PCR 體系檢測(cè)7 種不同種屬細(xì)菌中的ampC 基因。 6、利用標(biāo)記Cy3 的PCR 產(chǎn)物與寡核苷酸芯片雜交的技術(shù)建立檢測(cè)ampC 基因的方法。結(jié)果 1、在389 株革蘭陰性桿菌中,ampC 基因的陽(yáng)性率為:51.7%。在不同種屬的細(xì)菌中,ampC 基因的分布不一致:陰溝腸桿菌34 株(79.1%)、埃希氏菌屬93 株(79.5%)、克雷伯氏菌屬14 株(1.22%)、假單胞菌屬41 株(61.2%)和不動(dòng)桿菌屬14 株(50.0%)。 2、在201 株ampC 基因陽(yáng)性的菌株中,有34 株細(xì)菌產(chǎn)AmpC 酶,ampC 基因表達(dá)的總陽(yáng)性率為:16.9%。
[Abstract]:Improper use of antibiotics leads to the increasingly serious drug resistance of bacteria, which is a worldwide problem in clinical anti-infective therapy. One way to solve this problem is to target antibiotics based on the drug resistance of infected bacteria. At present, the detection of bacterial drug resistance still relies on the traditional drug sensitivity test, its biggest shortcoming is that the diagnosis cycle is long, the clinicians can not use reasonable drugs to the patients in the early stage of infection. Direct detection of bacterial drug resistance genes by gene chip technology can quickly and sensitively determine the drug resistance of bacteria and provide evidence for the rational use of antibiotics. Gram-negative bacilli are common pathogenic bacteria in clinic, and their treatment mainly depends on 尾-lactam antibiotics. Gram-negative bacilli tolerance to 尾 -lactam antibiotics is mainly caused by 尾 -lactamases produced by bacteria. There are many kinds of 尾 -lactamases, among which the more important ones are AmpC enzymes, such as ESBLs. AMPC enzymes belong to class I or Ambler C enzymes in Bush classification. The encoding gene is AMPC. We found that the ampC gene sequences of different species of bacteria were significantly different. Objective to analyze the relationship between the distribution of ampC resistance genes in clinical Gram-negative bacilli and the status of drug resistance, and to establish a method for the detection of multiplex PCR and oligonucleotide microarray for the detection of ampC resistance genes. Methods 1. The strains collected from the first affiliated Hospital of the third military Medical University and the Laboratory Department of Children's Hospital were isolated from October 2003 to May 2004. 2. PCR was used to detect the distribution of ampC gene in 389 Gram-negative bacilli. 3The method of cefxitin three-dimensional test was used to detect bacteria. The condition of producing AmpC enzyme. 4. Microscan walkaway-4.0 was used to identify the drug resistance of bacteria. 5. A multiplex PCR system was established to detect 7 different species. The method of detecting ampC gene was established by hybridization of PCR products labeled with Cy3 and oligonucleotide chip. Results 1. The positive rate of ampC gene in 389 Gram-negative bacilli was: 51.7%. The distribution of ampC gene was different among different species: Enterobacter cloacae 34 (79.1%), Escherichia coli 93 (79.5%), Klebsiella 14 (1.22%), Pseudomonas 41 (61.2%) and Acinetobacter 14 (50.0%). (2) among 201 ampC positive strains, The total positive rate of AmpC enzyme ampC gene expression in 34 strains of bacteria was: 16.9%.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R346
本文編號(hào)：2186088
[Abstract]:Improper use of antibiotics leads to the increasingly serious drug resistance of bacteria, which is a worldwide problem in clinical anti-infective therapy. One way to solve this problem is to target antibiotics based on the drug resistance of infected bacteria. At present, the detection of bacterial drug resistance still relies on the traditional drug sensitivity test, its biggest shortcoming is that the diagnosis cycle is long, the clinicians can not use reasonable drugs to the patients in the early stage of infection. Direct detection of bacterial drug resistance genes by gene chip technology can quickly and sensitively determine the drug resistance of bacteria and provide evidence for the rational use of antibiotics. Gram-negative bacilli are common pathogenic bacteria in clinic, and their treatment mainly depends on 尾-lactam antibiotics. Gram-negative bacilli tolerance to 尾 -lactam antibiotics is mainly caused by 尾 -lactamases produced by bacteria. There are many kinds of 尾 -lactamases, among which the more important ones are AmpC enzymes, such as ESBLs. AMPC enzymes belong to class I or Ambler C enzymes in Bush classification. The encoding gene is AMPC. We found that the ampC gene sequences of different species of bacteria were significantly different. Objective to analyze the relationship between the distribution of ampC resistance genes in clinical Gram-negative bacilli and the status of drug resistance, and to establish a method for the detection of multiplex PCR and oligonucleotide microarray for the detection of ampC resistance genes. Methods 1. The strains collected from the first affiliated Hospital of the third military Medical University and the Laboratory Department of Children's Hospital were isolated from October 2003 to May 2004. 2. PCR was used to detect the distribution of ampC gene in 389 Gram-negative bacilli. 3The method of cefxitin three-dimensional test was used to detect bacteria. The condition of producing AmpC enzyme. 4. Microscan walkaway-4.0 was used to identify the drug resistance of bacteria. 5. A multiplex PCR system was established to detect 7 different species. The method of detecting ampC gene was established by hybridization of PCR products labeled with Cy3 and oligonucleotide chip. Results 1. The positive rate of ampC gene in 389 Gram-negative bacilli was: 51.7%. The distribution of ampC gene was different among different species: Enterobacter cloacae 34 (79.1%), Escherichia coli 93 (79.5%), Klebsiella 14 (1.22%), Pseudomonas 41 (61.2%) and Acinetobacter 14 (50.0%). (2) among 201 ampC positive strains, The total positive rate of AmpC enzyme ampC gene expression in 34 strains of bacteria was: 16.9%.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R346
【引證文獻(xiàn)】
相關(guān)期刊論文 前1條
1 何昕;多麗波;;AmpC β-內(nèi)酰胺酶實(shí)驗(yàn)室檢測(cè)方法及進(jìn)展[J];中華醫(yī)院感染學(xué)雜志;2011年21期
，本文編號(hào)：2186088
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