Quantitative PCR (qPCR) or real-time PCR are used to amplify and quantify targeted DNA molecules simultaneously. The process involves amplification of one or more specific sequences in a DNA sample and at the same time, a fluorescent dye is included in the reaction mixture to provide real-time quantification. However, the said 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, our technology can intercalate DNA and selectively detect the cells that exhibit metabolic activity. Hence, it has the innate ability to detect inactivation under conditions that do not impact membrane permeability.
This is a novel molecular viability assay to selectively detect only the cells that exhibit metabolic activity. Current study has shown that coupled with qPCR, it can be used effectively to distinguish between active and nonactive gram-negative bacteria (P. aeruginosa PAO1) and gram-positive bacteria (E. faecalis v583). This is comparable to the effect of a current state-of-the art Propidium monoazide (PMA)-qPCR assay.
The bacteriological testing market was USD 9.58 billion in 2017, and is projected to reach USD 13.98 billion by 2022, at a compound annual growth rate of 7.8%. Culture methods require 4-5 days to obtain presumptive positive or negative results and can take up to 7 days, depending on the biochemical and serological confirmations. The current dyes used for viability PCR target cells with compromised membranes. Not all inactivation methods disrupt the cell membrane so not all nonviable cells have compromised membranes. As a result, the estimation of the viable bacteria is inaccurate and bacterial cells can remain visibly intact and impermeable to the dyes for hours or days after the viability is lost.