荧光PCR探针熔解分析法检测质粒介导ampC耐药基因的应用与评价

发布时间：2018-02-01 18:37

本文关键词： 质粒 AmpC酶聚合酶链反应肺炎克雷伯菌大肠埃希菌　出处：《中华医院感染学杂志》2016年02期 　论文类型：期刊论文

【摘要】：目的探讨多重荧光PCR-探针熔解分析法应用于临床分离大肠埃希菌和肺炎克雷伯菌中ampC耐药基因的检测方法,并评价该方法的临床应用价值。方法收集医院2009年7月-2010年6月的临床分离菌株,首先采用头孢西丁纸片法进行耐药表型筛选,再同时应用传统PCR技术与荧光PCR-探针熔解曲线法对临床分离菌株进行检测,并对ampC耐药基因扩增产物进行DNA测序。结果经头孢西丁纸片法筛选出176株对头孢西丁不敏感临床分离菌株,其中97株为肺炎克雷伯菌、79株为大肠埃希菌;在176株临床分离株中,荧光PCR-探针熔解分析法检测出36株ampC耐药基因阳性菌株,包括18株DHA型、12株CIT型和5株EBC型,还有1株肺炎克雷伯菌同时含有DHA型和EBC型;而传统PCR技术检出32株ampC耐药基因阳性,两个检测结果的符合率为97.7%;DNA测序后经BLAST比对,荧光PCR-探针熔解分析法检测ampC基因型与所检测目的基因型一致。结论荧光PCR-探针熔解分析法能高效检测出质粒介导ampC耐药基因,其方法简便、敏感性高、特异性强,具有良好的临床应用价值。
[Abstract]:Objective to investigate the application of multiplex fluorescence PCR- probe fusion assay in the detection of ampC resistance genes in clinical isolates of Escherichia coli and Klebsiella pneumoniae. The clinical application value of this method was evaluated. Methods the clinical isolates from July 2009 to June 2010 were collected and the resistant phenotypes were screened by cefoxitin disk method. At the same time, the traditional PCR technique and fluorescence PCR-probe melting curve method were used to detect the clinical isolates. The amplified products of ampC resistance gene were sequenced by DNA. Results 176 clinical isolates were screened by cefxitin disk method, 97 of which were Klebsiella pneumoniae. 79 strains were Escherichia coli. Among 176 clinical isolates, 36 ampC resistant gene positive strains were detected by fluorescence PCR- probe fusion analysis, including 18 DHA type 12 CIT and 5 EBC type. Another strain of Klebsiella pneumoniae contained both DHA and EBC types. 32 strains of ampC resistance genes were detected by traditional PCR technique, and the coincidence rate of the two results was 97.7%. DNA was sequenced and compared with BLAST. The detection of ampC genotypes by fluorescence PCR-probe fusion assay is consistent with that of the target genotypes. Conclusion fluorescence PCR-probe fusion assay can efficiently detect plasmide-mediated ampC resistance genes. The method is simple, sensitive and specific, and has good clinical application value.
【作者单位】：厦门大学附属第一医院检验科;厦门大学生命科学院生物医学科学系;厦门市中心血站;
【基金】：国家自然科学基金资助项目(81000762) 福建省自然科学基金资助项目(2013D002) 福建省卫生厅青年基金资助项目(2010-2-90) 厦门科技局基金资助项目(3502z20089003)
【分类号】：R446.5
【正文快照】： 3.厦门市中心血站,福建厦门361004)β-内酰胺酶是导致临床病原菌耐药最主要的耐药机制之一,C类β-内酰胺酶,即AmpC酶,不仅由染色体编码产生,20世纪90年代以后,质粒介导AmpC酶逐渐在肺炎克雷伯菌和大肠埃希菌等革兰阴性杆菌中被发现[1],由于耐药基因存在于质粒载体上,使ampC耐

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