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

Abstract

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.

Enterococcus faecalis: Its Role in Root Canal Treatment Failure and Current Concepts in Retreatment

“…In some cases, E. faecalis has been found as the only organism (pure culture) present in rootfilled teeth with periradicular lesions (4,28). The majority of these studies have been carried out using culturing techniques; however, polymerase chain reaction (PCR) is currently a more predictable method for detection of E. faecalis (34,35). This method proves to be faster, more sensitive, and more accurate than culturing methods (35).…”

Real-time Quantitative Polymerase Chain Reaction and Culture Analyses of Enterococcus faecalis in Root Canals

“…Each 100 l endodontic sample was thawed and centrifuged at room temperature for 5 min at 16,000 ϫ g. Cell pellets were resuspended in 50 l sterile nuclease-free water (Invitrogen), heated at 100°C for 5 minutes then cooled to room temperature. To each suspension was added 1 mg lysozyme, 150 g achromopeptidase, and 15 g mutanolysin to facilitate bacterial cell wall degradation (23). After incubation of 1 hour at 37°C, genomic DNA extractions were performed using the Wizard DNA Purification Kit (Promega, Madison, WI) according to the manufacturer's suggested protocol but scaled down to accommodate a 100 l sample.…”

Microbial Leakage of Gutta-percha and Resilon™ Root Canal Filling Material: A Comparative Study Using a new Homogeneous Assay for Sequence Detection

“…After 7 days, the root canal dressing was removed as follows: flushing with 5 mL 5% NaOCl at [40][41][42][43][44][45][46][47][48][49][50] C; K-Flexofile 20 ISO (Dentsply Maillefer, Ballaigues, Switzerland) 1 mm short of the working length (WL); flushing with 5 mL 10% EDTA; flushing with 5 mL 5% NaOCl at [40][41][42][43][44][45][46][47][48][49][50] C; K-Flexofile 20 ISO 1 mm short of the WL; flushing with 5 mL 10% EDTA; K-Flexofile 20 ISO 1 mm short of the WL; final flush with 5 mL 10% EDTA for 2 min.…”