High Through-put Sequencing Analysis Of Microbial 16 SrDNA In Chronic Pulpitis And Apical Periodontitis Root Canals

Objective:Pulp and periapical diseases are caused by various microorganism,the function and distribution of microorganism is an essential issue in the clinical researches.This experiment analysed 16 SrDNA microbial in root canals from chronic pulpitis and apical periodontitis by high through-put sequencing.The, microbial community of pulp and periapical infection,the relation between microorganism was discussed in the paper.This study will provide experiment facts to disease’s diagnoses,classify standard and the infection control methods during root canal therapy.Methods:121 cases diagonosed chronic pulpitis,chronic apical periodontitis with and without sinus tract were collected. Samples’DNA were extracted and double PCR amplification was applied.After the real-time fluorescence quantification,high through-put sequencing was applied.2 After the valid sequence optimized, the majorized sequence acquired and removed the non repetitive sequence. The sequence clustered in accordance with the 97% similarity and get representative sequences of OTU(Operational Taxonomic Uint) were collected. The result contrasted with Silva 119 databases and find out the species that had the most similar information and whose credibility was over 80%.The taxons of Phylum,Class,Order,Family,Genus,Specis were acquired.3 Alpha-diversity was analysed,based on the sequence similarity of OTU and so were the taxonomy of species, the analysis of Beta-diversity, significant difference analysis, correlation analysis of microorganisms.Results:1 There were 2 samples in the group of chronic pulpitis failed in DNA extraction.5 cases of chronic pulpitis,7 cases chronic apical periodontitis without sinus tract and 7 cases chronic apical periodontitis with sinus tract,were tested by high through-put sequencing.2 Contrasted with Sliva 119 databases,a total of 443320 OTUs at 97% same were obtained,covering 19 bacterial phyla,177 genus,159 species.3 Shannon index curve indicates satisfied coverage.Coverage of every sample was more than 99%. Rarefaction curve showed each samples in this study sequencing data was reliable.17 genus had high detection rate in sample total content, all of their content were over 1%,and a total of 65.36%.11 genus had 100% detection rate in samples,which was 44% in sample total content. The detection rate of Acinetobacter, Neisseria, Olsenella, Phyllobacterium, Bacillus, Streptococcus were significantly different(P<0.05) in the all classes. The detection rate of 7 bacterial genues were significantly different(P<0.05) in group of chronic pulpitis and chronic apical periodontitis without sinus tract.The detection rate of 54 bacterial genues were significantly different(P<0.05) in group of chronic pulpitis and chronic apical periodontitis with sinus tract. The detection rate of 7 bacterial genues were significantly different(P<0.05) in group of chronic apical periodontitis without and with sinus tract. Correlation analysis of microorganisms indicated that in the group of chronic pulpitis Acinetobacter,Phyllobacterium,Bacillus and other bacteria were symbiotic; Leptotrichia, Acinetobacter,Phyllobacterium,Bacillus and other bacteria were antagonistic. In the group of chronic apical periodontitis without sinus tract Acinetobacter,Lactobacillus,Phyllobacterium.Bacillus and other bacteria were symbiotic; Actinomyces.Dalister and other bacteria were antagonistic. In the group of chronic apical periodontitis with sinus tract Acinetobacter,Phyllobacterium.Bacillus and other bacteria were symbiotic; Phocaeicola,prevotella, Lactobacillus and other bacteria were antagonistic. Conclusion:A analysis of microorganism in root canals from chronic pulpitis and apical periodontitis by high through-put sequencing showed good coverage.The microorganisms of chronic pulpitis,chronic apical periodontitis without sinus tract, chronic apical periodontitis with sinus tract showed different microbial diversity.The group of chronic pulpitis,chronic apical periodontitis with or without sinus tract, bacteria has symbiosis and antagonism relation pattens.