应用分子开关识别23SrRNA基因突变检测肺炎支原体对大环内酯类抗生素的敏感性

发布时间：2018-03-15 01:33

  本文选题：肺炎支原体　切入点：大环内酯类抗生素　出处：《南华大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：检测自南华大学附属第一医院儿科住院患儿中分离培养的肺炎支原体（Mycoplasma pneumoniae, MP）临床株对大环内酯类抗生素的敏感性，用基于高保真聚合酶和3’硫化修饰引物的分子开关行PCR扩增，建立一种快速、准确、灵敏的检测MP23S rRNA基因突变的新方法，指导临床合理用药治疗MP感染。 方法：收集2010年~2012年南华大学附属第一医院儿科住院516例患儿咽拭子标本，用支原体SP4液体培养基进行分离培养，CM401琼脂培养基培养支原体菌落进行纯化，，并通过PCR进行筛选和进一步鉴定。采用微量稀释法检测5种常用大环内酯类抗生素（14元环抗生素红霉素、克拉霉素；15元环的阿奇霉素、交沙霉素和16元环的吉他霉素）对MP临床分离株的药物敏感性。并用高保真聚合酶和3’硫化修饰引物的分子开关行PCR扩增，检测是否存在MP23S rRNA2063、2064和2617三个热点突变，通过基因测序进一步确定是否存在点突变。分析点突变与大环内酯类抗生素敏感性之间的关系。 结果：516例呼吸道感染患儿咽拭子标本共分离培养出21株MP，分离阳性率为4.07%（21/516）；PCR扩增出49例阳性，检出率为9.50%（49/516）。21株MP中，仅2株大环内酯类抗生素敏感株，19株为高度耐药株，耐药率为90.5%（19/21），未检测出中度耐药株。5种大环内酯类抗生素中，MP对14元环的红霉素和克拉霉素耐药程度最高，其MIC≥128mg/L；对15元环的大环内酯类抗生素阿奇霉素和交沙霉素相对敏感，其中交沙霉素抗MP活性最高，其MIC≤4mg/L。用高保真聚合酶和3’硫化修饰引物的分子开关行PCR扩增，检测出17株发生了2063位点基因突变，2株发生2064位点基因突变，未检测出2617位点突变。用基因测序检测到17株MP发生A2063G的突变，2株发生A2064G的突变，未检测到2617位点突变，与分子开关的检测结果一致，而且2063、2064位点突变MP株均对大环内酯类药物高度耐药。 结论： 1.南华大学附属第一医院分离的MP株对大环内酯类抗生素高度耐药。 2.分子开关可识别23S rRNA基因突变、分析MP对大环内酯类抗生素的敏感性。
[Abstract]:Objective: to detect the sensitivity of Mycoplasma pneumoniae (MPN) strains isolated from pediatric hospitalized children in the first affiliated Hospital of South China University to macrolide antibiotics. PCR amplification was performed by using molecular switches based on high fidelity polymerase and 3'sulphide modified primer. A new rapid, accurate and sensitive method for detecting MP23S rRNA gene mutation was established to guide rational drug use in clinical treatment of MP infection. Methods: from 2010 to 2012, 516 pharyngeal swabs were collected in pediatrics department of the first affiliated Hospital of South China University. Mycoplasma mycoplasma colony was isolated and cultured on SP4 liquid medium. The microdilution method was used to detect erythromycin, azithromycin and azithromycin of five common macrolide antibiotics, erythromycin, clarithromycin and azithromycin. The drug sensitivity of josamycin and Guitamycin (16-member ring) to the clinical isolates of MP was detected by PCR amplification with high fidelity polymerase and 3'sulphide modified primer molecular switch to detect whether there were three hot spot mutations of MP23S rRNA2063C2064 and 2617. The relationship between point mutation and the susceptibility of macrolides to macrolides was further determined by gene sequencing. Results 21 strains of MPP were isolated from throat swabs of 516 children with respiratory tract infection. The positive rate was 4.07%. 49 cases were positive by PCR. The positive rate was 9.50%. Of the 21 strains of MP, only 2 strains of macrolide antibiotic sensitive strains were highly resistant. The drug resistance rate was 90.5% / 21%. Among the medium resistant strains of 5 macrolides, MP had the highest resistance to erythromycin and clarithromycin with MIC 鈮

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