本文選題：肺炎支原體　切入點(diǎn)：大環(huán)內(nèi)酯類抗生素　出處：《南華大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：檢測(cè)自南華大學(xué)附屬第一醫(yī)院兒科住院患兒中分離培養(yǎng)的肺炎支原體（Mycoplasma pneumoniae, MP）臨床株對(duì)大環(huán)內(nèi)酯類抗生素的敏感性，用基于高保真聚合酶和3’硫化修飾引物的分子開關(guān)行PCR擴(kuò)增，建立一種快速、準(zhǔn)確、靈敏的檢測(cè)MP23S rRNA基因突變的新方法，指導(dǎo)臨床合理用藥治療MP感染。 方法：收集2010年~2012年南華大學(xué)附屬第一醫(yī)院兒科住院516例患兒咽拭子標(biāo)本，用支原體SP4液體培養(yǎng)基進(jìn)行分離培養(yǎng)，CM401瓊脂培養(yǎng)基培養(yǎng)支原體菌落進(jìn)行純化，，并通過PCR進(jìn)行篩選和進(jìn)一步鑒定。采用微量稀釋法檢測(cè)5種常用大環(huán)內(nèi)酯類抗生素（14元環(huán)抗生素紅霉素、克拉霉素；15元環(huán)的阿奇霉素、交沙霉素和16元環(huán)的吉他霉素）對(duì)MP臨床分離株的藥物敏感性。并用高保真聚合酶和3’硫化修飾引物的分子開關(guān)行PCR擴(kuò)增，檢測(cè)是否存在MP23S rRNA2063、2064和2617三個(gè)熱點(diǎn)突變，通過基因測(cè)序進(jìn)一步確定是否存在點(diǎn)突變。分析點(diǎn)突變與大環(huán)內(nèi)酯類抗生素敏感性之間的關(guān)系。 結(jié)果：516例呼吸道感染患兒咽拭子標(biāo)本共分離培養(yǎng)出21株MP，分離陽性率為4.07%（21/516）；PCR擴(kuò)增出49例陽性，檢出率為9.50%（49/516）。21株MP中，僅2株大環(huán)內(nèi)酯類抗生素敏感株，19株為高度耐藥株，耐藥率為90.5%（19/21），未檢測(cè)出中度耐藥株。5種大環(huán)內(nèi)酯類抗生素中，MP對(duì)14元環(huán)的紅霉素和克拉霉素耐藥程度最高，其MIC≥128mg/L；對(duì)15元環(huán)的大環(huán)內(nèi)酯類抗生素阿奇霉素和交沙霉素相對(duì)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’硫化修飾引物的分子開關(guān)行PCR擴(kuò)增，檢測(cè)出17株發(fā)生了2063位點(diǎn)基因突變，2株發(fā)生2064位點(diǎn)基因突變，未檢測(cè)出2617位點(diǎn)突變。用基因測(cè)序檢測(cè)到17株MP發(fā)生A2063G的突變，2株發(fā)生A2064G的突變，未檢測(cè)到2617位點(diǎn)突變，與分子開關(guān)的檢測(cè)結(jié)果一致，而且2063、2064位點(diǎn)突變MP株均對(duì)大環(huán)內(nèi)酯類藥物高度耐藥。 結(jié)論： 1.南華大學(xué)附屬第一醫(yī)院分離的MP株對(duì)大環(huán)內(nèi)酯類抗生素高度耐藥。 2.分子開關(guān)可識(shí)別23S rRNA基因突變、分析MP對(duì)大環(huán)內(nèi)酯類抗生素的敏感性。
[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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