a new method to extract dental pulp dna application to universal detection of bacteria一种新方法来提取牙髓dna应用普遍的细菌检测.pdfVIP

A New Method to Extract Dental Pulp DNA: Application to Universal Detection of Bacteria ´ Lam Tran-Hung, Ny Tran-Thi, Gerard Aboudharam, Didier Raoult*, Michel Drancourt ´ ´ ´ ´ ´ ´ Unite des Rickettsies, CNRS UMR 6020, IFR 48, Faculte de Medecine, Universite de la Mediterranee, Marseille, France 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. Citation: Tra