TaqMan real-time PCR detection of Enterococcus faecium

To establish TaqMan real-time PCR assay of Enterococcus faecium and provide reliable technical basis for clinical application,we designed the primers and probe of Enterococcus faeciumaccording to ddl gene and detected the specificity in common pathogens and conditioned pathogens.The target gene was cloned to pMD18-T vector to build the standard curve of this method.We made the ascites simulation specimens with the concentrations from 1.6×100-1.6×108cfu/mL by 10series dilutions of Enterococcus faeciumstrain and mixing well with sterile ascites to evaluate the application in clinical lab.The target gene was positive in Enterococcus faeciumstandard strain and clinical isolates but negative in other pathogens and conditioned pathogens by TaqMan real-time PCR assay.According to the standard curve,we acquired the sensitivity of this method was 20copy/reaction.The stability evaluation indicated that the inter and intra-assay coefficients of variability were1.52%1.69% and 1.90%-3.91%,respectively.The sensitivity of TaqMan real-time PCR assay of the ascites simulation specimens was 1.6×102cfu/mL.In conclusion,the TaqMan real-time PCR assay developed in our study could be used in the detection of Enterococcusfaeciumin ascites specimens.