摘要
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 investigate the feasibility of high-resolution melting （HRM） analysis to detect gyrA gene mutation in Escherichia coll. Methods A total of 70 isolates of Escherichia coli were collected, and gyrA gene was amplified with conventional polymerase chain reaction （PCR）. Gene mutation was detected by sequence analysis. Specific primer was designed. The gyrA gene was amplified by fluorescence quantitation PCR. The mutation of gyrA gene was detected by amplifying product HRM curve and melting temperature （Tin） analysis. The result was compared with that of gene sequence analysis. Results gyrA gene mutation sequence analysis showed that 43 of 70 isolates occurred gyrA gene mutation. In these mutations, 5 types were ibund. HRM analysis can accurately distinguish between wild and mutant isolates. The detection accuracy was 98.6% （69/70）. The 42 of 43 mutant isolates were correctly detected, and different type gene mutations can be correctly typed. Tm analysis also showed the difference of wild and mutant isolates. Conclusions HRM analysis is accurate, simple, rapid, cheap and high-throughput, and it and would be a novel choice in analyzing drug resistance mechanism of Escherichia coli.
出处
《检验医学》
CAS
2015年第11期1138-1142,共5页
Laboratory Medicine
基金
江苏省连云港市卫生局科研项目(11014)
