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


RESULTS
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
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.
Tran-Hung
Lam
L
Unité des Rickettsies, CNRS UMR 6020, IFR 48, Faculté de Médecine, Université de la Méditerranée, Marseille, France.
Tran-Thi
Ny
N
Aboudharam
Gérard
G
Raoult
Didier
D
Drancourt
Michel
M
D016470:Bacteremia / Q000175:diagnosis* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification*; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000302:isolation & purification*; D012321:DNA-Directed RNA Polymerases; D003782:Dental Pulp / Q000378:metabolism* / Q000382:microbiology*; D003813:Dentistry / Q000379:methods; D005821:Genetic Techniques; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry*
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; C483832:rpoB protein, Streptococcus pneumoniae; D004247:DNA; D012321:DNA-Directed RNA Polymerases