A new method to extract dental pulp DNA: application to universal detection of bacteria

Abstract

Background: Dental pulp is used for PCR-based detection of DNA derived from host and bacteremic microorganims. Current protocols require odontology expertise for proper recovery of the dental pulp. Dental pulp specimen exposed to laboratory environment yields contaminants detected using universal 16S rDNA-based detection of bacteria.

Methodology/principal findings: We developed a new protocol by encasing decontaminated tooth into sterile resin, extracting DNA into the dental pulp chamber itself and decontaminating PCR reagents by filtration and double restriction enzyme digestion. Application to 16S rDNA-based detection of bacteria in 144 teeth collected in 86 healthy people yielded a unique sequence in only 14 teeth (9.7%) from 12 individuals (14%). Each individual yielded a unique 16S rDNA sequence in 1-2 teeth per individual. Negative controls remained negative. Bacterial identifications were all confirmed by amplification and sequencing of specific rpoB sequence.

Conclusions/significance: The new protocol prevented laboratory contamination of the dental pulp. It allowed the detection of bacteria responsible for dental pulp colonization from blood and periodontal tissue. Only 10% such samples contained 16S rDNA. It provides a new tool for the retrospective diagnostic of bacteremia by allowing the universal detection of bacterial DNA in animal and human, contemporary or ancient tooth. It could be further applied to identification of host DNA in forensic medicine and anthropology.

Figure 1. The original protocol developed in our study allows recovering the dental pulp and minimizes the risk of laboratory-acquired contamination of the specimen.

The tooth was encasted into sterile resin (1a) ; the apex was sterily sectioned (1b) to give access to the canal system (1c) ; solutions were injected (1d) ; after incubation, the tooth was put upside down into sterile tube (1e) and centrifuged (1f).

Figure 2. Amplification of a 286-bp 16S rDNA fragment in human dental pulp incorporting an original protocol for the decontamination of PCR reagents.

Lanes 1 and 8 feature molecular weight marker VI (molecular weight marker sizes in base-pairs are indicated in the left margin); lanes 4 and 5, negative controls; lanes 2, 3, 6 and 7, positive controls (C. burnetii DNA). Figure 2a, ineffective PCR reagent decontamination by using restriction enzymes alone. Figure 2b, effective PCR reagent decontamination by filtration followed by restriction enzyme digestion.

Figure 3. Decontamination of experimentally infected teeth.

Figure 3a, 16S rDNA partial amplification in 10 teeth exhibiting positive result in all teeth without decontamination: Lanes 1 and 12 feature molecular weight marker VI (molecular weight marker sizes in base-pairs are indicated in the left margin); lanes 2–11, teeth amplification. Figure 3b, 16S rDNA partial amplification in 10 teeth exhibiting negative result in all teeth with decontamination. Lanes 1 and 17 feature molecular weight marker VI (molecular weight marker sizes in base-pairs are indicated in the left margin); lanes 1,2,3,15 and 16, negative controls; lanes 4–14, teeth amplification.

Figure 4. Amplification of a 286-bp 16S rDNA fragment in human dental pulp incorporting an original protocol for the decontamination of PCR reagents.

Lanes 1 and 18 feature molecular weight marker VI (molecular weight marker sizes in base-pairs are indicated in the left margin); lanes 2 and 17, negative controls; lanes 3–16, dental pulp specimens exhibiting a positive amplification in lanes 6, 7, 12, 13, 14 and 16.