Detection and quantification of four periodontal pathogens using digital droplet-PCR

Abstract

In the treatment of periodontal disease, it can be of importance to know which bacteria are associated with the inflammatory response in individual patient. Microbiological testing is not common today as a routine test in periodontal treatment, often due to high costs and time consuming procedure. However, microbial diagnosis could improve the ability to identify patients at higher risk for developing periodontal disease, as well as monitor progression or remittance of diseases and, accordingly, choosing the appropriate course of treatment. Aims: The aim of this study is to develop a quick molecular method for simultaneous detection (multiplexing) and absolute quantification of four periodontal pathogens associated with periodontal disease in the same clinical sample by digital droplet PCR. Such a method would make the use of microbiological testing more effective and less expensive, and could encourage dentists to choose microbiological testing as a useful tool for prevention, diagnosis and treatment of periodontal diseases. Materials and method: Four designated periodontal pathogens were selected in this study. DNA was extracted from the four periodontal pathogens followed by amplification and quantification for performing a multiplex assay using droplet digital PCR (ddPCR). Results: A 4-plex system to detect four different periodontal pathogens was successfully achieved. In this assay, four periodontal bacteria divided into two groups were labelled with two fluorophores (FAM and HEX). The amount of primer utilized in the assay was adjusted so that each bacterium could be distinguished from the others on the basis of the fluorescence intensity. The designed assay managed to detect and quantify the four bacteria and recognize them separately or in groups. Conclusion: It seems the 4-plex assay developed herein is suitable for detection and quantification of periodontal pathogens, and the same assay can be used in other fields where accurate and reliable quantification and detection of multiple DNA-targets are needed. This 4-plex technique can make the workflow in detection and quantification of periodontal pathogens more effective by running just one sample in microbiological lab for several purposes.

Keywords: multiplexing, periodontitis, periodontal pathogens, digital droplet PCR