耐万古霉素肠球菌检测基因芯片方法的建立和验证
发布时间：2018-10-26 06:43
【摘要】：肠球菌属于革兰氏阳性球菌,它广泛分布于自然环境及人和动物的消化道内,其致病力较弱,只有在宿主组织寄殖,耐机体非特异及免疫防御机制,并引起病理改变才能导致感染。但是由于近年来在临床中广谱抗生素尤其是万古霉素的大量使用以及在使用中的不合理现象,导致了耐万古霉素肠球菌(VRE)的出现,从而引起了全世界范围内的广泛关注。近年来大量研究显示,由于VRE更容易引起院内感染及播散,其检测、鉴别与诊断也成为了医务工作者及研究者们所面临的重要问题。目前常用VRE的检测方法为细菌学方法,其使用耗时且无法准确辨别肠球菌的菌种类型;并且肠球菌属中不同种细菌对抗菌药物的耐药性存在差异,其也无法区分VRE的耐药基因。相对于药敏实验的不足,本研究选取了2种常见的肠球菌,旨在应用基因芯片检测技术研制可以区分VRE种类及耐药基因型的基因芯片,为深入开展基因芯片检测VRE更广泛应用于临床奠定基础。我们以16S rDNA基因作为检测靶片段,耐万古霉素特异性基因及肠球菌的特异性基因作为靶序列,根据GeneBank中查找到的2种常见肠球菌特异性基因序列(ddl)及万古霉素耐药基因(VanA,VanB),利用BLAST等比对软件,设计与合成用于检测肠球菌的特异性基因及耐药基因的引物和探针,制备耐万古霉素肠球菌检测基因芯片。利用多重不对称PCR法扩增样品中的特异性基因与耐药基因片段,标记产物与基因芯片上的探针杂交,经清洗、化学发光法显色后进行结果分析。在优化的多重PCR体系、杂交反应和化学发光法检测条件下,评价芯片的特异性、灵敏性和重复性等性能指标,并应用于临床样本的检测。最后本研究所选取的靶耐药基因和肠球菌特异性基因为耐万古霉素A基因(VanA)、耐万古霉素B基因(VanB)、肠球菌特异性基因(ddl),16S rDNA基因作为内对照,共筛选出1对通用引物、4对特异性引物和1条细菌通用探针、4条特异性检测探针。确定了五重PCR体系。确定了每种探针的cutoff值。检测耐万古霉素肠球菌的灵敏度为103cfu/mL,批间和批内变异系数CV值均小于15%。对40例临床样本进行检测,结果与常规培养法培养结果基本一致。因此综上所述本研究建立的耐万古霉素肠球菌基因芯片检测方法具有高灵敏性、高特异性且高通量的优点,基本能够区分肠球菌类型及相关耐药的基因型。该方法可为临床中耐万古霉素肠球菌的检测、个体化选择用药、监测预后及院内感染提供了更新的实验方法,其结果可靠,并降低了操作难度及成本,对于减少资源浪费和避免抗生素带来更多不良反应的风险有重要意义。
[Abstract]:Enterococcus belongs to Gram-positive cocci. It is widely distributed in the natural environment and the digestive tract of human and animal. Its pathogenicity is weak. And cause pathological changes to cause infection. However, in recent years, the widespread use and unreasonable use of broad spectrum antibiotics, especially vancomycin, has led to the emergence of vancomycin resistant Enterococcus (VRE), which has attracted worldwide attention. In recent years, a large number of studies have shown that because VRE is more likely to cause nosocomial infection and spread, its detection, identification and diagnosis have become an important problem for medical workers and researchers. At present, the commonly used VRE detection method is bacteriological method, which is time-consuming and can not accurately identify the type of Enterococcus, and the resistance of different strains of Enterococcus to antimicrobial agents is different, and it can not distinguish the drug-resistant genes of VRE. Compared with the deficiency of drug sensitivity test, two common enterococci were selected in this study. The purpose of this study was to develop a gene chip that could distinguish VRE species from drug resistant genotypes by using gene chip detection technology. It lays a foundation for further application of gene chip detection of VRE in clinical practice. We used 16s rDNA gene as target fragment, vancomycin-resistant specific gene and Enterococcus specific gene as target sequence. According to (ddl) and vancomycin resistant gene (VanA,VanB), two common enterococci specific gene sequences were found in GeneBank. BLAST et al. Primers and probes were designed and synthesized to detect the specific genes and drug resistance genes of Enterococcus enterococcus, and the vancomycin resistant Enterococcus microarray was prepared. The specific gene and drug resistance gene fragments were amplified by multiple asymmetric PCR method. The labeled products were hybridized with probes on the gene chip. The results were analyzed by cleaning and chemiluminescence method. In the optimized multiplex PCR system, hybridization reaction and chemiluminescence assay, the specificity, sensitivity and repeatability of the chip were evaluated and applied to the detection of clinical samples. Finally, the target resistance gene and Enterococcus specific gene were selected as internal control because vancomycin A gene, (VanA), gene, vancomycin B gene, (VanB), gene, Enterococcus specific gene (ddl), 16s rDNA gene, were selected as internal control. A pair of universal primers, four pairs of specific primers, one universal bacterial probe and four specific detection probes were screened. The five-fold PCR system is determined. The cutoff value of each probe was determined. The sensitivity of detection of vancomycin resistant Enterococcus was 103 cfur / mL, and the coefficient of variation (CV) was less than 15%. The clinical samples of 40 cases were detected and the results were consistent with the results of conventional culture method. In conclusion, the vancomycin resistant Enterococcus gene chip assay has the advantages of high sensitivity, high specificity and high throughput, and can basically distinguish Enterococcus and related drug resistant genotypes. This method can provide a new experimental method for the detection of vancomycin resistant Enterococcus, individualized drug selection, prognosis monitoring and nosocomial infection. The results are reliable, and the operation difficulty and cost are reduced. It is important to reduce the waste of resources and avoid the risk of more adverse reactions caused by antibiotics.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5
本文编号：2294887
[Abstract]:Enterococcus belongs to Gram-positive cocci. It is widely distributed in the natural environment and the digestive tract of human and animal. Its pathogenicity is weak. And cause pathological changes to cause infection. However, in recent years, the widespread use and unreasonable use of broad spectrum antibiotics, especially vancomycin, has led to the emergence of vancomycin resistant Enterococcus (VRE), which has attracted worldwide attention. In recent years, a large number of studies have shown that because VRE is more likely to cause nosocomial infection and spread, its detection, identification and diagnosis have become an important problem for medical workers and researchers. At present, the commonly used VRE detection method is bacteriological method, which is time-consuming and can not accurately identify the type of Enterococcus, and the resistance of different strains of Enterococcus to antimicrobial agents is different, and it can not distinguish the drug-resistant genes of VRE. Compared with the deficiency of drug sensitivity test, two common enterococci were selected in this study. The purpose of this study was to develop a gene chip that could distinguish VRE species from drug resistant genotypes by using gene chip detection technology. It lays a foundation for further application of gene chip detection of VRE in clinical practice. We used 16s rDNA gene as target fragment, vancomycin-resistant specific gene and Enterococcus specific gene as target sequence. According to (ddl) and vancomycin resistant gene (VanA,VanB), two common enterococci specific gene sequences were found in GeneBank. BLAST et al. Primers and probes were designed and synthesized to detect the specific genes and drug resistance genes of Enterococcus enterococcus, and the vancomycin resistant Enterococcus microarray was prepared. The specific gene and drug resistance gene fragments were amplified by multiple asymmetric PCR method. The labeled products were hybridized with probes on the gene chip. The results were analyzed by cleaning and chemiluminescence method. In the optimized multiplex PCR system, hybridization reaction and chemiluminescence assay, the specificity, sensitivity and repeatability of the chip were evaluated and applied to the detection of clinical samples. Finally, the target resistance gene and Enterococcus specific gene were selected as internal control because vancomycin A gene, (VanA), gene, vancomycin B gene, (VanB), gene, Enterococcus specific gene (ddl), 16s rDNA gene, were selected as internal control. A pair of universal primers, four pairs of specific primers, one universal bacterial probe and four specific detection probes were screened. The five-fold PCR system is determined. The cutoff value of each probe was determined. The sensitivity of detection of vancomycin resistant Enterococcus was 103 cfur / mL, and the coefficient of variation (CV) was less than 15%. The clinical samples of 40 cases were detected and the results were consistent with the results of conventional culture method. In conclusion, the vancomycin resistant Enterococcus gene chip assay has the advantages of high sensitivity, high specificity and high throughput, and can basically distinguish Enterococcus and related drug resistant genotypes. This method can provide a new experimental method for the detection of vancomycin resistant Enterococcus, individualized drug selection, prognosis monitoring and nosocomial infection. The results are reliable, and the operation difficulty and cost are reduced. It is important to reduce the waste of resources and avoid the risk of more adverse reactions caused by antibiotics.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5
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相关期刊论文 前2条
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