Rapid Multiplex Real-Time PCR Method for the Detection and Quantification of Selected Cariogenic and Periodontal Bacteria

Abstract

Dental caries and periodontal diseases are associated with a shift from symbiotic microbiota to dysbiosis. The aim of our study was to develop a rapid, sensitive, and economical method for the identification and quantification of selected cariogenic and periodontal oral bacteria. Original protocols were designed for three real-time multiplex PCR assays to detect and quantify the ratio of 10 bacterial species associated with dental caries ("cariogenic" complex) or periodontal diseases (red complex, orange complex, and Aggregatibacter actinomycetemcomitans). A total number of 60 samples from 30 children aged 2-6 years with severe early childhood caries and gingivitis were tested. In multiplex assays, the quantification of total bacterial (TB) content for cariogenic bacteria and red complex to eliminate differences in quantities caused by specimen collection was included. The mean counts for the TB load and that of ten evaluated specimens corresponded to previously published results. We found a significant difference between the microbial compositions obtained from the area of control and the affected teeth (p < 0.05). Based on this comprehensive microbiological examination, the risk of dental caries or periodontal inflammation may be determined. The test could also be used as a tool for behavioral intervention and thus prevention of the above-mentioned diseases.

Keywords: dental caries; dysbiosis; oral microbiome; periodontitis; risk factors determination.

Figure 1

Abundance of bacterial species among the tested interdental site samples. The counts are reported on a log₁₀ scale. Blue boxes correspond to the subtotals for species of cariogenic bacteria. Red and orange boxes correspond to the subtotals for species of the Socransky red and orange complexes.

Figure 2

Correlation plots between the counts of bacteria and their percentage content in samples. Exceptional values in Lactobacillus spp. and A. actinomycetemcomitans were omitted from the analysis. Black dots correspond to individual analyzed samples and dotted line shows trend line on a scatter plot.