Abstract
BackgroundDental 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 FindingsWe 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/SignificanceThe 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.
Highlights
Dental pulp is a specialized conjunctive tissue occupying the dental pulp cavity of the tooth
Molecular detection of bacteria in specimens can be achieved by the amplification and sequencing of highly specific targets
Universal molecular targets such as the 16S rDNA offer the opportunity to detect most of bacterial species but are subjected to non-specific amplification due to contaminant DNA from prelaboratory and laboratory sources
Summary
Dental pulp is a specialized conjunctive tissue occupying the dental pulp cavity of the tooth. Dental pulp has been used as a source of host DNA for genotyping buried individuals based on sequencing the DQA1 gene of the HLA region on chromosome 6 [17], STR loci [18] and hypervariable region 1 of the mitochondrial control region [19] In patients, it allowed for the posthumous molecular diagnosis of inherited diseases including Duchenne muscular dystrophy [20] and the Rett syndrome [21]. 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. It could be further applied to identification of host DNA in forensic medicine and anthropology
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