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

Clinical value of fluorescent PCR-probe melting analysis in detection of plasmid-mediated AmpC β-lactamase genes

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 explore the clinical value of multiplex real-time PCR-probe melting analysis in detection of ampC drug resistance gene in clinical isolates of Escherichia coli and Klebsiella pneumoniae.METHODS The clinical isolates were collected from Jul 2009 to Jun 2010;the drug resistance phenotypes were screened by using cefoxitin disk agar diffusion method,the clinical isolates were detected by means of the traditional PCR technique and fluorescent PCR-probe melting curve method,and the DNA sequencing was performed for the amplified products of the ampC drug resistance gene.RESULTS Totally 176 clinical isolates that were not sensitive to cefoxitin were screened out by using cefoxitin disk method,including 97 K.pneumoniaeisolates and 79 E.coli isolates.Of the 176 clinical isolates,36 were detected positive for the ampC drug resistance gene by using the fluorescent PCR-probe melting analysis method,including 18 strains positive for DHA,12 strains positive for CIT,5strains positive for EBC,and 1strain positive for both DHAand EBC;while 32 strains were detected positive for the ampC drug resistance gene by the traditional PCR technique,the coincidence rate of the two detection results was97.7%;the DNA sequencing analysis showed that by contrast to BLAST,the ampCdrug resistance gene detected by the fluorescent PCR-probe melting analysis method was consistent with the target genotype.CONCLUSIONThe fluorescent PCR-probe melting analysis method can effectively detect the plasmid-mediated ampCdrug resistance gene,it is simple,highly sensitive and specific and worthy to be promoted in hospitals.