Use of Ethidium Monoazide and PCR in Combination for Quantification of Viable and Dead Cells in Complex Samples

Abstract

The distinction between viable and dead cells is a major issue in many aspects of biological research. We have developed a novel concept for quantification of viable and dead cells in complex samples. The viable/dead stain ethidium monoazide (EMA) is used in combination with realtime PCR to inhibit amplification of DNA from dead cells that have taken up EMA (Fig. 1). Viable/dead determinations are key issues in many aspects of biological research. The current technologies addressing this important issue have severely limited application ranges (4,5,14,18,19). There are for instance no approaches enabling accurate viable/dead quantifications in mixed cell populations (2, 13).Real-time PCR is the most widely applied technology for direct quantification of cells in mixed samples. Real-time PCR is increasingly being used for direct detection and quantification of pathogens in foods and environmental or clinical samples. Still, a major obstacle with PCR diagnostics is how to distinguish between DNA from viable and dead cells. Intact DNA can be present although the organisms are dead. This is particularly relevant for pathogens subjected to killing treatments such as disinfections or antibiotics. Even greater challenges are encountered with organisms such as Campylobacter jejuni that have specific growth requirements and may enter a state where it is viable and infectious but not cultivable. The lack of viable/dead differentiation has been a serious limitation for the implementation of DNA diagnostics in routine applications (15,19,22).We have recently used ethidium monoazide (EMA)-PCR for qualitative DNA-based viable/dead differentiation of bacteria in pure monoculture models (21). Viable/dead analyses of pure monocultures are not new or novel. A wide range of different approaches exist (2,5,10,12,25,26). Methods for direct quantitative analyses of complex samples, however, are still lacking. Solving these analytical problems would be a major technological breakthrough. We discovered during the work with the monoculture models that EMA-PCR has this potential. Thus, the aim of the present work was to use EMA-PCR to show that it is possible to develop quantitative assays for specific viable and dead bacteria in complex samples with mixed bacterial populations. We developed the assay for the major food-borne pathogenic bacterium C. jejuni due to the apparent need for new viable/dead diagnostics of this bacterium. The conditions analyzed include detection in mixed and natural samples, survival in foods, and after disinfection and antibiotic treatments. This knowledge is crucial both for diagnostics and in the control of C. jejuni.A dynamic range of more than 4 log 10 was obtained for the EMA-PCR viable/dead assay. We were able to reliably quantify the fraction of viable C. jejuni under all conditions tested, including complex samples with mixed populations. This is to our knowledge the first time that quantitative viable/dead information has been obtained from specific bacteria in mixed populations. C. jejuni was used as...

Development of a quantitative PCR method to differentiate between viable and nonviable bacteria in environmental water samples

“…Samples were treated as described by Rudi et al (2005a) with slight modifications. Briefly, 1 ml of sample was centrifuged at 13,000×g for 10 min, the supernatant removed and the pellet resuspended in 1 ml of EMA at concentrations of 0, 1, 2, 3, 5, and/or 7.5 μg/ml followed by incubation in the dark at room temperature for 5 min.…”

Molecular viability testing of viable but non‐culturable bacteria induced by antibiotic exposure

“…Despite the advantages of culture‐independent methods, the limitation of molecular assessment (especially for DNA‐based methods) is the possible overestimation of viable cell densities because DNA can persist for an extended period after cell death in environments (Rudi et al ., 2005). Recently, DNA‐intercalating dyes such as ethidium monoazide (EMA) and propidium monoazide (PMA) have been proposed as useful molecular methods to quantify intact bacterial cells in environments (Nogva et al ., 2003; Rudi et al ., 2005; Nocker et al ., 2006). PMA or EMA with PCR technique has been widely used to assess cell viability in water, soil, air and food (Rogers et al ., 2008; Bae and Wuertz, 2009; Miotto et al ., 2012; Kaushik and Balasubramanian, 2013; Li and Chen, 2013).…”

Viable quantitative PCR for assessing the response of Candida albicans to antifungal treatment

“…Selective nucleic acid intercalating dyes, such as EMA and PMA, represent one of the most successful recent approaches to detect viable cells (as defined by an intact cell membrane) by PCR (Fittipaldi et al 2012) and have been effectively evaluated in different microorganisms Rudi et al 2005;van Frankenhuyzen et al 2011). As both dyes have similar structures, comparable results might be expected; however, some studies have showed differences (Andorrà et al 2010;Cawthorn et al 2008;Chang et al 2010;Nam et al 2011;Nocker and Camper 2009;Pan and Breidt 2007;Wagner et al 2008).…”

“…Photolysis of EMA and PMA converts the azide group into a highly reactive nitrene radical, which can react with any organic molecule in its proximity, including DNA. In this bound state, DNA cannot be amplified by PCR (Nocker and Camper 2009;Rudi et al 2005). This promising analytical approach is still in development and needs to be investigated further (Fittipaldi et al 2012).…”