Validation of a multiplex qPCR assay for the identification and quantification of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis: In vitro and subgingival plaque samples

Abstract

ObjectiveTo validate a multiplex qPCR (m-qPCR) assay for the simultaneous identification and quantification of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis in subgingival samples. Material and methodsIn vitro samples: DNA combinations of A. actinomycetemcomitans and P. gingivalis in similar or different concentrations were prepared. qPCR and m-qPCR were performed using the same primers and hydrolysis probes specific for 16SrRNA genes. Results were analyzed using intra-class (ICCs) and Lin’s correlation coefficients (r) based on quantification cycle (Cq) values. Subgingival plaque samples: a cross-sectional study analyzing subgingival plaque samples harvested from periodontally-healthy and chronic periodontitis patients. Samples were processed by either qPCR or m-qPCR targeting both bacteria. Sensitivity, specificity, predictive values and Lińs correlation coefficients (r) were calculated using CFU/mL as primary outcome. ResultsIn vitro samples: m-qPCR yielded a good reproducibility (coefficients of variation around 1% and ICCs > 0.99) for both bacterial species. m-qPCR achieved detection limits and specificity similar to qPCR. An excellent concordance (r = 0.99) was observed between m-qPCR and qPCR for A. actinomycetemcomitans and P. gingivalis without statistical significant differences between both methods Subgingival plaque samples: a high sensitivity (above 80%) and specificity (100%) was obtained with the m-qPCR for both bacteria. The m-qPCR yielded a good concordance in Cq values, showing a good level of agreement between qPCR and m-qPCR. ConclusionThe tested m-qPCR method was successful in the simultaneous quantification of A. actinomycetemcomitans and P. gingivalis, with a high degree of sensitivity and specificity on subgingival plaque samples.