Epidemiological Survey Of Tooth Exogenous Pigmentation And Microbial Flora Analysis Of Supragingival Plaque And Saliva Of Primary Dentition

[Objectives] To know the prevalence of exogenous pigmentation of primary dentition.16S rRNA454high-throughput barcoded pyrosequencing was used to examine and compaire the oral microbiota in supragingival plaques and un-stimulative saliva from the primary dentition, then the correlation of oral microbiota with exogenous pigmentation was analised.[Methods] Based on large sample principles, cluster sampling method was used in this study. The representative samples were selected from5public urban kindergartens and7public suburban kindergartens of Kunming,1273children from3to5years old were invited to participate in the survey. Each child was clinically examined using portable equipments,1273samples were collected. Epidata3.1and SPSS17.0were used for data compilation and statistical analysis.454high-throughput pyrosequencing was used to analyze bacterial diversity of oral microbiota in un-stimulative saliva and supragingival plaque. All samples were from66children of primary dentition, the children were divided into two groups,26were normal with no caries and no pigmentation,40were pure pigmentation with no caries. Dental supragingival plaque and un-stimulative saliva were collected, total bacterial DNA were extracted and the V1-V3region of16S rRNA were amplified with specific primers. PCR products were detected by agarose gel electrophoresis, the purpose DNA fragments were purificated and collected. The concentration of DNA fragments was examined with the Qubit2.0fluorometer. Equal dose of DNA fragments was mixed for sequencing by454pyrosequencing. The dedicated software was used for data processing and analysis. [Results](1) The prevalence of exogenous pigmentation of3-5years old primary dentition was9.7%(boys’ was10.1%, girls’9.4%) in Kunming. It was11.9%(boys’ was13.0%, girls’ was10.9%) in urban areas. It was7.0%(boys’ was6.6%, girls’was7.3%) in suburban areas. The prevalence of exogenous pigmentation of the children in urban areas was higher than that of suburban children’s (p<0.05). It was higher of the urban boys’than that of the suburban boys’(p<0.05). There was no statistical significance with gender and that of in urban and suburban areas. There was no statistical significance between urban and suburban areas with girls’.(2)707411high quality sequencing reads were obtained on bacterial16S rRNA gene’ V1-V3region from132supragingival plaque and unstimulated saliva samples. The average length of the sequences was282bp. The average number of sequence reads was5359per sample. According to16S rRNA sequence database, a total of55182OTUS at3%difference were obtained, covering14phylums and170genera.(3) The sample species abundance distribution map and the diversity of Alpha diversity dilution curve indicated good bacteria coverage in this study.(4) There were34genera with a significant difference in exogenous pigmentation plaque compared with healthy plaque (p<0.05),10genera were dominant in exogenous pigmentation plaque.17genera with a significant difference in exogenous pigmentation saliva compared with healthy saliva (p<0.05),13genera were dominant in exogenous pigmentation saliva.[Conclusions](1)The prevalence of exogenous pigmentation of3-5years old primary dentition was higher than that of previous reports, it was higher in urban areas.(2) In this study, there was a good bacteria coverage by454pyrosequencing analysis of supragingival plaque and unstimulated salivary microorganisms of primary teeth.(3) There were different microbial compositions with different parts in oral cavity.(4) The higher composition genera in supragingival plaque and unstimulated salivary of exogenous pigmentation, may be associated with exogenous pigmentation. The association needs to be studied by further researches.