Quantification of Bacterial DNA from Infected Human Root Canals Using qPCR and DAPI after Disinfection with Established and Novel Irrigation Protocols

Abstract

The removal of bacterial infections within the root canal system is still a challenge. Therefore, the cleansing effect of established and new irrigation-protocols (IP) containing silver diamine fluoride (SDF) 3.8% on the whole root canal system was analyzed using quantitative PCR (qPCR) and 4′,6-diamidino-phenylindole-(DAPI)-staining. Extracted human premolars were instrumented up to F2 (ProTaper Gold) under NaCl 0.9% irrigation and incubated with Enterococcus faecalis for 42 days. Subsequently, different ultrasonically agitated IP were applied to the roots: control (no irrigation), 1. NaOCl 3%, EDTA 20%, CHX 2%, 2. NaOCl 3%, EDTA 20%, 3. NaOCl 3%, EDTA 20%, SDF 3.8%, 4. SDF 3.8%, and 5. NaCl 0.9%. One half of the root was investigated fluorescent-microscopically with DAPI. The other half was grinded in a cryogenic mill and the bacterial DNA was quantified with qPCR. The qPCR results showed a statistically significant reduction of bacteria after the application of IP 1, 2, and 3 compared to the control group. While IP 4 lead to a bacterial reduction which was not significant, IP 5 showed no reduction. These data corresponded with DAPI staining. With qPCR a new molecular-biological method for the investigation of the complete root canal system was implemented. The novel IP 3 had an equally good cleansing effect as the already established IP.