PCR identification of endodontic pathogens and DNA quantification in samples from teeth with posttreatment apical periodontitis

Abstract

Aim: The aim of this clinical study was to quantify the concentration of DNA and to detect selected bacterial species from samples of infected root-fi lled teeth with post-treatment apical periodontitis after removal of gutta-percha (S1), after chemo-mechanical preparation at the fi rst appointment (S2), 5 days after the canal was fi lled with sterile physiological solution (S3), after reinstrumentation at the second appointment (S4), and 14 days after an intracanal dressing was placed at the third appointment (S5). Methods: Fifteen root-fi lled teeth were selected. Removal of gutta-percha was performed using the crown-down technique. Chemo-mechanical preparation was performed with hand fi les associated with 2% chlorhexidine gel. An intracanal dressing based on Ca(OH)2 was used. DNA was extracted from the samples and 14 endodontic 16S rDNA species-specifi c primers were tested. The concentration of DNA was quantifi ed using a NanoDropTM 2000 spectrophotometer. Results: Bacteria were present in all cases at all sampling times, as revealed by a universal primer. DNA was isolated from all samples, with an average concentration of 4.24 ± 2.9 ng/µL (S1), 3.39 ± 1.54 ng/µL (S2), 4.0 ± 1.94 ng/µL (S3), 2.66 ± 0.98 ng/µL (S4) and 3.97 ± 2.32 ng/µL (S5). Parvimonas micra and Enterococcus faecalis (S1), P. micra (S2), Porphyromonas endodontalis and E. faecalis (S3), E. faecalis and Prevotella nigrescens (S4/S5) were the species most frequently deteced. DNA concentration reductions were detected between S3 and S4 (p = 0.0256), whereas an increase was found between S4 and S5. Conclusion: A wide variety of bacterial species was detected in root-fi lled teeth with post-treatment apical periodontitis. Moreover, the use of an intracanal dressing was unable to further reduce the concentration of bacterial DNA.