Analysis of viable vs. dead Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis using selective quantitative real-time PCR with propidium monoazide

Abstract

Background and Objectives: One of the major disadvantages of DNA-based microbial diagnostics is their inability to differentiate DNA between viable and dead microorganisms, which could be important when studying etiologically relevant pathogens. The aim of this investigation was to optimize a method forthe selective detection and quantification of only viable Aggregatibacter actino-mycetemcomitans and Porphyromonas gingivalis cells by combining quantitative real-time polymerase chain reaction (qPCR) and propidium monoazide (PMA). Material and Methods: Three different concentrations of PMA (10, 50 or 100lM)were added to suspensions of 106(CFU)/mL of viable/dead A. actinomycetem-comitans and P. gingivaliscells. After DNA isolation, qPCR was carried out using specific primers and probes for the tested bacteria. PMA was further tested with different mixtures containing varying ratios of viable and dead cells. The efficacyof PMA to detect viable/dead cells was tested by analysis of variance. Results: For these specific bacterial pathogens, 100lMPMA resulted in a signif-icant reduction of qPCR amplification with dead cells (106CFU/mL), while with viable cells no significant inhibition was detected. PMA was also effectivein detecting selectively viable cells by qPCR detection, when mixtures of varying ratios of viable and dead bacteria were used. Conclusions: This study demonstrated the efficiency of PMA for differentiating viable and dea dA. actinomycetemcomitans and P. gingivaliscells. This method of PMA-qPCR may be useful for monitoring new antimicrobial strategies and for assessing the pathogenic potential ofA. actinomycetemcomitansandP. gingi-valisin different oral conditions when using molecular diagnostic methods.