Validation of a quantitative real-time PCR assay and comparison with fluorescence microscopy and selective agar plate counting for species-specific quantification of an in vitro subgingival biofilm model

Background and Objective Subgingival biofilms are the prime etiological factor of periodontal disease. Owing to their complex polymicrobial nature, quantification of individual bacterial species within the biofilm for research and diagnostic purposes can be methodologically challenging. The aims of this study were to establish a quantitative real-time PCR (qPCR) assay to quantify the bacteria used in our 10-species in vitro subgingival' biofilm model and to compare the quantitative outcome with fluorescence microscopy and colony-forming unit (CFU) counts on selective agar plates. Material and Methods The 10 species included in the in vitro biofilm were Streptococcus oralis, Streptococcus anginosus, Veillonella dispar, Fusobacterium nucleatum, Treponema denticola, Tannerella forsythia, Actinomyces oris, Campylobacter rectus, Porphyromonas gingivalis and Prevotella intermedia. The numbers of each species were quantified at two time points using qPCR, microscopy counting following fluorescence in-situ hybridization (FISH) or immunofluorescence staining, and counting of CFUs after growth on selective agar plates. Results All 10 species were successfully quantified using qPCR and FISH or immunofluorescence, and the eight species culturable on selective agar plates were also quantified by counting the numbers of CFUs after growth on selective agar. In early biofilm cultures, all methods showed a significant correlation, although the absolute numbers differed between methods. In late biofilm cultures, measurements obtained using qPCR and FISH or immunofluorescence, but not by CFU counts, maintained significant correlation. CFU counts yielded lower values than did measurements made using the other two methods. Conclusion Quantitative PCR and epifluorescence microscopy can be easily combined with each other to determine species-specific bacterial numbers within biofilms. However, conventional bacterial cultures cannot be as efficiently combined using these molecular detection methods. This may be crucial in designing and selecting appropriate clinical diagnostic methods for subgingival biofilm samples.