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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。
Objective To investigate the detection of ampC resistance gene in Escherichia coli and Klebsiella pneumoniae by multiplex fluorescence PCR-probe melting analysis and to evaluate the clinical value of this method. Methods The clinical isolates from July 2009 to June 2010 in the hospital were collected. The resistant strains were screened by cefoxitin disk diffusion method. At the same time, the clinical isolates were separated by traditional PCR and fluorescent PCR-probe melting curve The strains were tested, and ampC resistance gene amplification products for DNA sequencing. Results A total of 176 strains of cefoxitin-insensitive clinical isolates were screened by cefoxitin disk method, of which 97 were Klebsiella pneumoniae and 79 were Escherichia coli. Of 176 clinical isolates, fluorescence PCR 36 strains of ampC resistance gene were detected by probe melting analysis, including 18 strains of DHA, 12 strains of CIT and 5 strains of EBC, and 1 strain of Klebsiella pneumoniae containing both DHA and EBC. However, 32 strains of ampC resistance gene were detected by traditional PCR technique, and the coincidence rate of the two test results was 97.7%. After DNA sequencing, the genotypes of ampC and the target genes detected by fluorescence PCR-probe fusion assay The same type. Conclusion Fluorescent PCR-probe melting assay can detect plasmid-mediated ampC resistance gene efficiently. The method is simple, sensitive, specific and has good clinical value.