Novel approaches toward preferential detection of viable cells using nucleic acid amplification techniques

Abstract

This article elaborates on possible future directions for microbial viability assessment using nucleic acid-modifying compounds in combination with DNA- (and potentially RNA-) amplification technologies. Bacteria were traditionally considered viable when they could be cultured, whereas today's viability concept is based on the presence of some form of metabolic activity, responsiveness, RNA transcripts that tend to degrade rapidly after cell death, or of an intact membrane. The latter criterion was the focus of recent approaches to limit detection to intact cells using ethidium monoazide or propidium monoazide. Membrane integrity must, however, be considered as a very conservative criterion for microbial viability. The new concept presented here aims at limiting nucleic acid-based detection to cells with an active metabolism, which might be a more appropriate viability criterion. To selectively detect only cells with metabolic and respiratory activity (while excluding inactive dead cells from detection), we suggest the use of ‘activity-labile compounds’. In addition to their potential usefulness for viability assessment, these new compounds could also be beneficial for selectively amplifying nucleic acids of cells that have metabolic activities of interest. This preferential detection of microorganisms with certain metabolic capabilities is referred to as ‘molecular enrichment’ in distinction to ‘growth enrichment’.