Evaluating the newly developed dye, DyeTox13 Green C-2 Azide, and comparing it with existing EMA and PMA for the differentiation of viable and nonviable bacteria.

Abstract

PCR-based methods for enumerating bacteria cells could lead to an overestimation of viable cells due to the amplification of DNA from dead cells. To overcome this disadvantage, DNA intercalating dyes such as Ethidium monoazide (EMA) or Propidium monoazide (PMA) combined with qPCR have been considered promising alternative methods to discriminate between viable and nonviable cells. The drawback of those DNA intercalating dyes, however, could not assess the physiological states of cells. Thus, the objective of this study was to develop a novel molecular viability assay to selectively detect only the cells that exhibit metabolic activity. This study's results showed that DyeTox13 Green C-2 Azide dye coupled with qPCR can be used effectively to distinguish between active and nonactive gram-negative bacteria (P. aeruginosa PAO1) and gram-positive bacteria (E. faecalis v583). In this study, we evaluated the usefulness of the DyeTox13 Green C-2 Azide-qPCR assay for selectively amplifying nucleic acids of microorganisms that have metabolic activities. We conclude that the use of DyeTox13 Green C-2 Azide-qPCR is a promising alternative for discriminating active cells from nonactive cells but the dye's performance might be dependent on the kind of bacterial species.