利用寡核苷酸芯片检测耐药基因ampC的初步研究
发布时间：2018-08-16 13:13
【摘要】：抗生素的使用不当导致细菌耐药性的日益严重是临床抗感染治疗面临的世界性难题。解决这一问题的方法之一是根据感染细菌的耐药性有针对性地使用抗生素。目前对细菌耐药性的检测仍然依赖传统的药敏实验,其最大的缺点是诊断周期长,临床医生无法在感染早期对患者合理用药。通过基因芯片技术直接检测细菌耐药基因,可以快速、敏感地确定细菌的耐药性,为合理使用抗生素提供依据。革兰阴性杆菌是目前临床常见的致病菌,其治疗主要依赖β-内酰胺类抗生素。革兰阴性杆菌耐受β-内酰胺类抗生素主要由细菌产生的β-内酰胺酶引起。β-内酰胺酶种类繁多,其中较为重要的包括AmpC 酶、ESBLs 等。AmpC 酶属于Bush 分类中的I 类酶或Ambler C 类酶,其编码基因是ampC。我们发现,不同种属的细菌的ampC 基因序列显著不同。 目的分析临床革兰阴性杆菌中ampC 耐药结构基因的分布与耐药状况的关系;在此基础上建立起利用多重PCR 及寡核苷酸芯片技术检测ampC 耐药结构基因的方法。 方法 1、标本菌株收集来源于第三军医大学第一附属医院及儿童医院检验科2003 年10月至2004 年5 月临床分离的菌株。 2、用PCR 检测389 株革兰阴性杆菌中ampC 基因的分布。 3、应用头孢西丁三维实验的方法检测细菌产AmpC 酶的状况。 4、应用microscan walkaway-4.0 鉴定细菌的耐药性。 5、建立多重PCR 体系检测7 种不同种属细菌中的ampC 基因。 6、利用标记Cy3 的PCR 产物与寡核苷酸芯片杂交的技术建立检测ampC 基因的方法。结果 1、在389 株革兰阴性杆菌中,ampC 基因的阳性率为:51.7%。在不同种属的细菌中,ampC 基因的分布不一致:阴沟肠杆菌34 株(79.1%)、埃希氏菌属93 株(79.5%)、克雷伯氏菌属14 株(1.22%)、假单胞菌属41 株(61.2%)和不动杆菌属14 株(50.0%)。 2、在201 株ampC 基因阳性的菌株中,有34 株细菌产AmpC 酶,ampC 基因表达的总阳性率为: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%.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346
本文编号：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%.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346
【引证文献】
相关期刊论文 前1条
1 何昕;多丽波;;AmpC β-内酰胺酶实验室检测方法及进展[J];中华医院感染学杂志;2011年21期
，本文编号：2186088
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