摘要目的 建立高分辨率熔解曲线法筛选含VEB-3型基因菌株.方法 不重复收集2003年1-12月革兰阴性杆菌产超广谱β-内酰胺酶(ESBL)菌株292株,用酚-氯仿法抽提基因组DNA;首先以基因组DNA为模板,PCR扩增VEB基因;挑选阳性菌株34株再PCR扩增包含168位点的一段约110 bp的基因片段,高分辨率熔解曲线(high resolution melt,HRM)分析后测序验证.结果 VEB-3亚型与其他亚型基因通过HRM分析具有显著的差别;测序结果与HRM分型结果一致.结论 应用HRM技术可以准确快速筛选含VEB-3型基因菌株.

abstractsObjective To establish a new method, applying high resolution melt, to discriminate the VEB-3 hypotype from the clinical gram negative isolates. Methods From January to December 2003,292 consecutive and non-repetitive gram-negative bacteria producing VEB extended spectrum β-lactamase (ESBL) were collected. Extract the DNA of clinical gram negative isolates with phenol-chloroform. PCR was performed to amplify the VEB gene with the DNA being template. After that, we amplify the fragment of VEB gene containing the position 168. Then we detect the high resolution melt curve and analyze them. At last, we analyze the results of sequence and high resolution melt( HRM ). Results VEB-1 and VEB-3 gene are markedly different through HRM analysis. Conclusion It is accurately and quickly for us to identify the VEB-3 from other hypotype through the technology of HRM.