發(fā)布時間：2018-03-22 19:28

  本文選題：多重耐藥鮑曼不動桿菌　切入點：耐藥基因　出處：《南方醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：背景與目的耐藥基因blaoxa-23、blaAde-Bblaint、1及blaaIsCR-1在多重耐藥鮑曼不動桿菌耐藥機制中發(fā)揮著重要的作用,臨床微生物實驗室如能快速、準確檢測這4種耐藥基因攜帶情況,不僅可以幫助臨床醫(yī)生了解菌株攜帶耐藥基因情況,指導臨床合理用藥,還可以指導醫(yī)院感染控制部門針對攜帶不同耐藥基因鮑曼不動桿菌感染患者選擇不同隔離方式,避免院內感染的暴發(fā)。本研究期望利用單管多重PCR及高分辨熔解曲線分析技術,建立一種可同時快速檢測多重耐藥鮑曼不動桿菌耐藥基因blaoxa-23、blaAde-B、blaint-及blaISCR-1的反應體系,并應用于臨床鮑曼不動桿菌分離株多重耐藥性檢測。研究方法用Primer Premier 5.0軟件針對每種耐藥基因設計2對不同引物,第一對引物用于初篩試驗,第二對引物用于建立多重PCR及高分辨熔解曲線分析技術。陽性菌株作為模板,將針對4種耐藥基因的第二對引物全部加入到一個PCR反應管中,查看多重PCR是否可擴增出全部的目標產物,高分辨熔解曲線分析技術是否可檢測出所有目標產物的Tm值。應用成功構建的多重PCR及高分辨熔解曲線分析技術,檢測某綜合醫(yī)院檢驗科2014年1月至12月分離到的部分多重耐藥鮑曼不動桿菌耐藥基因blaoxa-23、blaAde-B、blaint-1 及blaISCR-1 攜帶情況。研究結果4個不同多重PCR反應體系均可擴增出4種PCR產物,產物大小分別為139 bp、390bp、234bp及187bp。4種不同多重PCR反應體系擴增產物進行高分辨熔解曲線,每個多重PCR反應體系均可見4個在不同位置的峰,4個峰對應的Tm值分別為79.5 ℃、82.4 ℃、87.1 ℃及90.3 ℃,多重PCR擴增出的4種產物Tm值與常規(guī)HRM PCR分別檢測耐藥基因blaoxa-23、blaAde-B、blaint-1及blaIscR-1實際Tm值相同,說明構建的多重PCR可成功擴增出4種目標基因。采用成功建立的多重PCR及高分辨熔解曲線分析分析技術檢測79株多重耐藥鮑曼不動桿菌4種耐藥基因攜帶情況,73株菌同時攜帶有耐藥基因blaoxa-23及blaAde-B,1株攜帶有blaint-1,2株同時攜帶有blaint-1及blaIscR-1,3株同時攜帶有blaAde-B、blaint-1及blaISCR-1,未發(fā)現同時攜帶有4種耐藥基因的菌株。結論1.本研究利用多重PCR及高分辨熔解曲線分析技術成功建立了一個可快速檢測耐藥基因blaoxa-23、blaAde-B、blaint-1及blaISCR-1的反應體系,與傳統耐藥基因檢測檢測方法相比具有快速、高通量、成本較低以及減少污染的優(yōu)勢。2.臨床分離多重耐藥鮑曼不動桿菌主要攜帶blaoxa-23及blaAde-B,雖然攜帶blaint1及blaISCR-1菌株較少,但這部分菌株在抗菌藥物耐藥基因傳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，

本文編號：1650052