A protocol for polymerase chain reaction detection of Enterococcus faecalis and Enterococcus faecium from the root canal

Abstract

Aim: The present study was set up to develop a protocol for detection of Enterococcus faecalis and Enterococcus faecium from the root canal.

Methodology: A collection of type strains and clinical isolates ol E. faecalis and faecium was used. Specific polymerase chain reaction (PCR) primers targeted against the 16S/23S rDNA intergenic region were used and PCR reactions were set up. PCR products were run on TBE-agarose gel and analysed. The sensitivity of the PCR systems was studied using serial dilutions of (i) bacterial DNA and (ii) bacterial cells from E. faecalis. The specificity of the identification was tested against closely related species.

Results: All strains of E. faecalis and E. faecium produced identical amplicon profiles composed of two major bands corresponding to sizes of 320 and 420 bp. When amplifying DNA of higher purity, a third band of 600 bp became evident as well. Closely related species demonstrated single bands of various sizes and were easily distinguished from enterococci. The detection level of DNA from serial dilutions of DNA was 10(-13) g. The DNA extraction protocol from bacterial cell suspensions resulted in a detection level of 10 bacterial cells per sample. CONCLUSIONS The present study demonstrated a good potential for using PCR technology in the detection of F. faecalis and E. faecium from root canal samples. With a high specificity the methodology was able to detect 10 cells of E. faecalis.