荧光PCR法检测原料乳中大肠埃希氏菌喹诺酮类的耐药基因

Determination of quinolones-resistance genes in raw milk-derived Escherichia coli real-time fluorescence PCR

中文关键词:  大肠埃希氏菌  喹诺酮类抗菌药  耐药基因  双重荧光PCR

英文关键词:Escherichia coli  quinolones  drug resistance genes  duplex fluorescence PCR

中文摘要:

      目的 建立大肠埃希氏菌喹诺酮类耐药基因的TaqMan荧光聚合酶链反应(polymerase chain reaction, PCR)检测方法, 为原料乳中耐药大肠埃希氏菌的监测提供技术方案。方法 利用Chelex 100法快速获取细菌基因组DNA, 对大肠埃希氏菌aac(6’)-Ib-cr和qepA基因的序列进行分析, 利用Primer Express 3.0软件设计特异性引物和探针, 建立单重和双重TaqMan荧光PCR方法。通过耐药基因阳性菌株和原料乳中分离的大肠埃希氏菌菌株, 对方法的特异性和灵敏度进行评估。结果 建立的aac(6’)-Ib-cr和qepA基因的单重和双重荧光PCR反应均有特异性曲线扩增, 方法灵敏度为104 CFU/mL。北京地区52份原料乳中共分离出32株大肠埃希氏菌, 利用建立的双重荧光PCR方法检测aac(6’)-Ib-cr和qepA基因为阴性, 和环丙沙星肉汤稀释法药敏结果一致。结论 本方法针对aac(6’)-Ib-cr和qepA基因建立的TaqMan荧光PCR检测方法特异性强、灵敏度高, 可用于原料乳中大肠埃希氏菌常见喹诺酮类耐药基因的快速检测。

英文摘要:

      Objective To establish TaqMan real-time fluorescence PCR method for the detection of quinolones-resistance genes in raw milk-derived Escherichia coli, and to provide monitoring alternatives to monitor drug-resistant Escherichia coli in raw milk. Methods Chelex 100 method was used to extract bacterial genomic DNA. Based on the analysis of aac(6’)-Ib-cr and qepA gene sequences of E.coli, the specific primers and probes were designed by software Premier Express 3.0, and the single TaqMan real-time fluorescence PCR and duplex TaqMan real-time fluorescence PCR methods were established. The specificity and sensitivity of the method were evaluated by the drug-resistant gene-positive strains and the Escherichia coli strains isolated from raw milk. Results The aac(6’)-Ib-cr and qepA gene were amplified by single/duplex fluorescence PCR reactions, the sensitivity of the method was 104 CFU/mL. The duplex fluorescent PCR results of aac(6’)-Ib-cr and qepA gene in the 32 strains of Escherichia coli isolated from 52 raw milk samples in Beijing were negative, which was consistent with the results of ciprofloxacin broth dilution method. Conclusion The established Taqman fluorescence PCR detection method is highly specific and sensitive, and it can be used for rapid detection of quinolones-resistance genes of Escherichia coli in raw milk.