采用多重PCR及高分辨熔解曲线分析技术快速检测多重耐药鲍曼不动杆菌4种耐药基因及其临床应用的研究

发布时间：2018-03-22 19:28

  本文选题：多重耐药鲍曼不动杆菌　切入点：耐药基因　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景与目的耐药基因blaoxa-23、blaAde-Bblaint、1及blaaIsCR-1在多重耐药鲍曼不动杆菌耐药机制中发挥着重要的作用,临床微生物实验室如能快速、准确检测这4种耐药基因携带情况,不仅可以帮助临床医生了解菌株携带耐药基因情况,指导临床合理用药,还可以指导医院感染控制部门针对携带不同耐药基因鲍曼不动杆菌感染患者选择不同隔离方式,避免院内感染的暴发。本研究期望利用单管多重PCR及高分辨熔解曲线分析技术,建立一种可同时快速检测多重耐药鲍曼不动杆菌耐药基因blaoxa-23、blaAde-B、blaint-及blaISCR-1的反应体系,并应用于临床鲍曼不动杆菌分离株多重耐药性检测。研究方法用Primer Premier 5.0软件针对每种耐药基因设计2对不同引物,第一对引物用于初筛试验,第二对引物用于建立多重PCR及高分辨熔解曲线分析技术。阳性菌株作为模板,将针对4种耐药基因的第二对引物全部加入到一个PCR反应管中,查看多重PCR是否可扩增出全部的目标产物,高分辨熔解曲线分析技术是否可检测出所有目标产物的Tm值。应用成功构建的多重PCR及高分辨熔解曲线分析技术,检测某综合医院检验科2014年1月至12月分离到的部分多重耐药鲍曼不动杆菌耐药基因blaoxa-23、blaAde-B、blaint-1 及blaISCR-1 携带情况。研究结果4个不同多重PCR反应体系均可扩增出4种PCR产物,产物大小分别为139 bp、390bp、234bp及187bp。4种不同多重PCR反应体系扩增产物进行高分辨熔解曲线,每个多重PCR反应体系均可见4个在不同位置的峰,4个峰对应的Tm值分别为79.5 ℃、82.4 ℃、87.1 ℃及90.3 ℃,多重PCR扩增出的4种产物Tm值与常规HRM PCR分别检测耐药基因blaoxa-23、blaAde-B、blaint-1及blaIscR-1实际Tm值相同,说明构建的多重PCR可成功扩增出4种目标基因。采用成功建立的多重PCR及高分辨熔解曲线分析分析技术检测79株多重耐药鲍曼不动杆菌4种耐药基因携带情况,73株菌同时携带有耐药基因blaoxa-23及blaAde-B,1株携带有blaint-1,2株同时携带有blaint-1及blaIscR-1,3株同时携带有blaAde-B、blaint-1及blaISCR-1,未发现同时携带有4种耐药基因的菌株。结论1.本研究利用多重PCR及高分辨熔解曲线分析技术成功建立了一个可快速检测耐药基因blaoxa-23、blaAde-B、blaint-1及blaISCR-1的反应体系,与传统耐药基因检测检测方法相比具有快速、高通量、成本较低以及减少污染的优势。2.临床分离多重耐药鲍曼不动杆菌主要携带blaoxa-23及blaAde-B,虽然携带blaint1及blaISCR-1菌株较少,但这部分菌株在抗菌药物耐药基因传播中起到了十分重要的作用。
[Abstract]:Background & objective the drug resistance genes blaoxa-23 blaAde-Bblaint1 and blaaIsCR-1 play an important role in the mechanism of multidrug resistance of Acinetobacter baumannii. If the clinical microorganism laboratory can detect these four drug-resistant genes quickly and accurately, It can not only help clinicians understand the drug resistance genes of the strains, but also guide the hospital infection control departments to select different isolation methods for patients with Acinetobacter baumannii infection with different drug resistance genes. In order to avoid the outbreak of nosocomial infection, the aim of this study was to establish a reaction system for rapid detection of multidrug resistant Acinetobacter baumannii resistance gene blaoxa-23 blaAde-Bde-Bde blaint- and blaISCR-1 by using single-tube multiplex PCR and high-resolution fusion curve analysis. Primer Premier 5.0 software was used to design 2 pairs of different primers for each drug resistance gene, and the first pair of primers was used to screen the drug resistance of Acinetobacter baumannii isolates. The second pair of primers was used to establish multiplex PCR and high-resolution fusion curve analysis technique. The positive strain was used as template, and the second pair of primers for the four resistant genes were all added to one PCR reaction tube. See if the multiple PCR can amplify all the target products, and whether the high resolution melting curve analysis technique can detect the TM value of all the target products. Using the successfully constructed multiplex PCR and the high resolution melting curve analysis technology, The resistance genes of Acinetobacter baumannii (Acinetobacter baumannii) gene blaoxa-23 blaAde-Bnblaint-1 and blaISCR-1 were isolated from laboratory department of a general hospital from January to December 2014. The results showed that four PCR products could be amplified from four different multiplex PCR reaction systems. The product size was 139bpn390bpc234bp and the high resolution melting curve of the amplified products from different multiplex PCR reaction system of 187bp.4 species was carried out. There were four peaks at different positions in each multiplex PCR reaction system, and the TM values of the four peaks were 79.5 鈩，

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