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目的探讨高分辨率溶解曲线(HRM)分析法在检测大肠埃希菌gyr A基因突变中的可行性。方法收集临床非重复分离的大肠埃希菌70株,常规聚合酶链反应(PCR)扩增大肠埃希菌的gyr A基因,采用序列分析法检测gyr A基因的变异情况。设计特异性引物,采用荧光定量PCR扩增这70株大肠埃希菌株的gyr A基因,通过对扩增产物进行HRM分析和熔解温度(Tm)值测定确定gyr A基因变异情况。将检测结果与序列分析法检测结果进行比较。结果基因测序显示70株大肠埃希菌中有43株gyr A基因发生了突变,突变类型分为5型。HRM分析法可以准确区分野生株和突变株,准确率达98.6%(69/70),正确检测出了43株突变菌株中的42株,且对不同的突变类型准确分型。Tm分析显示野生株和突变株之间以及各种突变型之间有明显差异。结论HRM技术具有准确性高、简单、快速、成本低廉、高通量等优点,可以作为检测大肠埃希氏菌gyr A基因突变耐药机制的新选择。
Objective To explore the feasibility of high resolution resolution curve (HRM) analysis in the detection of gyr A gene mutation in Escherichia coli. Methods Seventy isolates of Escherichia coli isolated from clinical isolates were collected. The gyr A gene of Escherichia coli was amplified by conventional polymerase chain reaction (PCR) and the gyr A gene was detected by sequence analysis. The specific primers were designed and the gyr A gene of 70 Escherichia coli strains was amplified by fluorescence quantitative PCR. The variation of gyr A gene was determined by HRM analysis and melting temperature (Tm) determination of the amplified product. The test results and sequence analysis test results were compared. Results The gene sequencing revealed that 43 strains of gyr A gene were mutated in 70 strains of Escherichia coli. The mutation types were classified as type 5. The accuracy of HRM analysis was 98.6% (69/70) for distinguishing wild and mutant strains, 42 strains were correctly detected in 43 mutant strains, and the exact mutation types were accurately identified. Tm analysis revealed significant differences between wild-type and mutant strains and among various mutant types. Conclusion The HRM technique has the advantages of high accuracy, simplicity, rapidity, low cost and high throughput. It can be used as a new choice to detect the mechanism of drug resistance in gyr A gene of Escherichia coli.