Progress in understanding preferential detection of live cells using viability dyes in combination with DNA amplification.
The ideal scenario in most applications of microbial diagnostics is that only viable cells are detected. Bacteria were traditionally considered viable when they could be cultured, whereas today's viability concept tends to be alternatively based on the presence of some form of metabolic activity, a positive energy status, responsiveness, detection of RNA transcripts that tend to degrade rapidly after cell death, or of an intact membrane. The latter criterion, although conservative, was the focus of one of the most successful recent approaches to detect viable cells in combination with DNA amplification techniques. The technology is based on sample treatment with the photoactivatable, and cell membrane impermeant, nucleic acid intercalating dyes ethidium monoazide (EMA) or propidium monoazide (PMA) followed by light exposure prior to extraction of DNA and amplification. Light activation of DNA-bound dye molecules results in irreversible DNA modification and subsequent inhibition of its amplification. Sample pretreatment with viability dyes has so far been mainly used in combination with PCR (leading to the term viability PCR, v-PCR), and increasingly with isothermal amplification method. The principle is not limited to bacteria, but has also successfully been applied to fungi, protozoa and viruses. Despite the success of the method, some practical limitations have been identified, especially when applied to environmental samples. In part they can be minimized by choice of experimental parameters and conditions adequate for a particular sample. This review summarizes current knowledge and presents aspects which are important when designing experiments employing viability dyes.