耐万古霉素肠球菌检测基因芯片方法的建立和验证

Gene chip technique in detection of vancomycin-resistant Enterococcus gene

目的基于基因芯片技术建立一种能快速甄别粪肠球菌、屎肠球菌及万古霉素耐药基因的检测方法。方法根据Gen Bank中查找的2种常见肠球菌特异性基因序列(ddl)及万古霉素耐药基因(van A,van B)序列,设计与合成用于检测肠球菌的特异性基因及耐药基因的引物和探针,制备耐万古霉素肠球菌(VRE)检测基因芯片。利用多重不对称PCR法扩增样品中的特异性基因与耐药基因片段,标记产物与基因芯片上的探针杂交,经清洗、化学发光法显色后进行结果分析。在优化的多重PCR体系、杂交反应和化学发光法检测条件下,评价芯片的特异性、灵敏性和重复性。结果共筛选出1对通用引物、4对特异性引物和1条细菌通用探针、4条特异性检测探针。该芯片具有良好的特异性和重复性,灵敏性可达103CFU/ml。10例临床分离株样本的芯片检测结果与药敏实验基本一致(8/10)。结论初步建立了检测VRE的基因芯片方法,利用此方法可以甄别2种VRE种类并检测其耐药基因。

Objective To develop a detection method based on the technology of gene chips which can quickly distinguish genes of Enterococcus faecalis,E. faecium and vancomycin resistance. Methods Based on the specific gene（ ddl） sequences of two types of Enterococcus from Gen Bank,oligonucleotide probes which could detect and distinguish special genes and drug resistance genes（ van A,van B） of Enterococcus were designed and compounded. Then,the probes were dotted to modified slide. The target DNA fragments of vancomycin-resistant Enterococcus（ VRE） were labeled with biotin by multiple PCR amplification,and then hybridized with oligonucleotide probes on slide. The results were analyzed by portable imager. The multiple PCR system,hybridization reaction and condition of the chemiluminescence method were optimized before the specificity,sensitivity and reproducibility of the chip were evaluated. Results One universal primer,four specific primers,one universal probe and four specific probes were selected. This gene chip was demonstrated of high specificity and repeatability. The detection sensitivity was 103 CFU / ml. The gene chip detection results of 10 clinical samples were basically consistent with the drug sensitivity test（ 8 /10）. Conclusion A gene chip technique for the detection of VRE is established successfully. It is possible to distinguish the type of VRE and detect the genetic phenotypes of drug resistance by gene chip technique.