Study of TaqMan real time PCR for detection of Enterococcus faecalis

Objective To establish a TaqMan real-time PCR assay to detect Enterococcus faecalis and provide reliable technical basis for its clinical application. Methods We designed the primers and probe for ddl gene of E. faecalis. The specificity of the assay was tested by using 39 common pathogens and opportunistic pathogens. In order to evaluate the sensitivity, the standard curve was built by using the target gene which was cloned into pMD18-T plasmid. We made the blood simulative specimens with the concentrations of E. faecalis from 3.9×100－3.9×108cfu/ml by 10 series dilutions of E. faecalis strain and mixing well with blood to evaluate the application in clinical sample detection. Results The target gene was positive in E.faecalis reference strains and clinical isolates but negative in other pathogens by this TaqMan real-time PCR assay. The standard curve showed that 20 copy E.faecalis per reaction could be detected by this assay. The sensitivity of this assay for blood simulative specimens was 3.9×103cfu/ml. The stability evaluation indicated that the coefficient of variation was from 0.99% to 1.84%. Conclusion The TaqMan real-time PCR assay established in our study could be used in the detection of E.faecalis in blood specimens.