PCR primer sets for surveying oral synergistetes diversity

Abstract

Objectives: Bacterial taxa belonging to the phylum Synergistetes commonly inhabit the human oral cavity. They are predominantly detected within periodontal niches; however, their distributions within asymptomatic versus diseased sites remains relatively poorly characterized.To design PCR primers that ‘specifically’ target the 16S rRNA gene of oral Synergistetes, and determine their efficacy for investigating the diversity of Synergistetes taxa present within periodontal niches Methods: Chinese subjects with chronic periodontitis (n=1); aggressive periodontitis (n=1), chronic periodontitis and peri-implantitis (n=2); and periodontitis-free controls (n=1); were recruited with informed consent. Subgingival plaque samples were collected from individual diseased and disease-free tooth and implant sites (where applicable), and DNA was purified (n=14). Two pairs of PCR primers that respectively targeted the 16S rRNA genes of oral cluster A and B Synergistetes were designed and tested in silico. Both primer sets amplified ca. 650 bp amplicons, and were used in a PCR-based approach to construct plasmid clone libraries of Synergistetes taxa present within each sample. Approximately 50-60 clones per Synergistetes-positive sample were sequenced, and data were analyzed using various bioinformatic software tools. Results: A total of 378 non-chimeric Synergistetes 16S rRNA sequences were identified, which were classified within 29 operational taxonomic units (OTUs, 99% sequence identity). Both primer sets had 100% specificity for Synergistetes taxa, with OTU coverage ranging from 94-100% (rarefaction curve analysis). Oral Synergistetes cluster A predominated (27 OTUs). Only one subgingival site (chronic periodontitis) yielded oral Synergistetes cluster B taxa (two OTUs, including Pyramidobacter piscolens). Individual peri-implantitis and periodontitis tooth sites harboured diverse Synergistetes communities (range: 2-12 OTUs). Synergistetes taxa were detected in asymptomatic sites in periodontitis subjects, but no Synegistetes were detected in the healthy control. Conclusions: Both PCR primer sets are effective at amplifying oral cluster A and B Synergistetes taxa, respectively, with good coverage and excellent specificity. Support Funding Agency/Grant Number: Research Grants Council (RGC) of Hong Kong, General Research Fund (#780713)