金属β内酰胺酶基因bla_(NDM)重组酶聚合酶扩增方法的建立

Establishment of recombinase polymerase amplification assay for drug resistance gene bla_(NDM) coding metallo-beta-lactamase

目的建立一种基于重组酶聚合酶(recombinase polymerase amplification,RPA)扩增方法金属β内酰胺酶基因bla_(NDM)的快速检测方法,为广泛耐药菌基因类型的确定提供技术支持。方法遵照重组酶聚合酶扩增引物和探针设计原则,以bla_(NDM)基因特异性序列为靶基因,设计相应的RPA扩增引物和探针。取梯度稀释已知拷贝数的DNA模板和致病菌核酸样品进行各扩增体系的RPA扩增实验,分析扩增体系的敏感性、特异性和重复性特征以便筛选和评价bla_(NDM)基因的RPA扩增体系。结果 3组扩增体系的引物和探针能够有效的扩增靶基因,检出限达到2×10~2copys/反应,与其他细菌样品无非特异性扩增反应,能够在2～7 min内实现靶基因有效扩增。结论建立了金属β内酰胺酶基因bla_(NDM)的重组酶聚合酶扩增体系,该反应迅速,检出限较低,具有较高的特异性,适用于耐药菌的现场检测。

OBJECTIVE To establish a recombinase polymerase amplification (RPA)method for extensively resistant pathogen screening and rapid detection in the field for rapid amplification of the metallo-beta-lactamase gene blaNDM.METHODS Specific conservative sequence had been selected as target genes by sequence comparative analysis. The primers and probes for RPA assays were designed according to the principle of RPA amplification requirements.A descending gradient diluted template genes were used for RPA detection to determine the sensitivity.Reference tempXates of other resistant types of bacteria were used to analysis the specificity of amplification reaction systems.The amplification reaction systems were also conducted repeatedly for verifying the repeatability.RESULTS Three of the RPA reaction systems could effectively amplify the target genes,the sensitivities reach 2~102 copies.No one cross reaction existed with the other drug-resistant bacteria DNA.All the reactions can be completed between two to seven minutes.CONCLUSION The RPA assays of the metallo-beta-lactamase gene blaNDM are established,which may amply target genes fast and have a lower detection limit,and be potentially useful for in field pathogens detection.