Selective covalent binding of an ethidium analog to mitochondrial DNA with production of petite mutants in yeast by photoaffinity labeling

134

mentioning

confidence: 99%

Viability PCR, a Culture-Independent Method for Rapid and Selective Quantification of Viable Legionella pneumophila Cells in Environmental Water Samples

Delgado‐Viscogliosi

Solignac

Delattre

2009

134

“…Following intercalation into the DNA of those cells, EMA can be covalently linked by photoactivation with high yields, up to 75% (3). Photolysis of EMA using visible light (maximum absorbance at 460 nm) produces a nitrene that can form a covalent linkage to DNA and other molecules (5,6,9). The unbound EMA, which remains free in solution, is simultaneously inactivated by reacting with water molecules (6).…”

mentioning

confidence: 99%

Selective Removal of DNA from Dead Cells of Mixed Bacterial Communities by Use of Ethidium Monoazide

Nocker¹,

Camper

2006

286

210

The distinction between viable and dead bacterial cells poses a major challenge in microbial diagnostics. Due to the persistence of DNA in the environment after cells have lost viability, DNA-based quantification methods overestimate the number of viable cells in mixed populations or even lead to false-positive results in the absence of viable cells. On the other hand, RNA-based diagnostic methods, which circumvent this problem, are technically demanding and suffer from some drawbacks. A promising and easy-to-use alternative utilizing the DNA-intercalating dye ethidium monoazide bromide (EMA) was published recently. This chemical is known to penetrate only into "dead" cells with compromised cell membrane integrity. Subsequent photoinduced crosslinking was reported to inhibit PCR amplification of DNA from dead cells. We provide evidence here that in addition to inhibition of amplification, most of the DNA from dead cells is actually lost during the DNA extraction procedure, probably together with cell debris which goes into the pellet fraction. Exposure of bacteria to increasing stress and higher proportions of dead cells in defined populations led to increasing loss of genomic DNA. Experiments were performed using Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium as model pathogens and using real-time PCR for their quantification. Results showed that EMA treatment of mixed populations of these two species provides a valuable tool for selective removal of DNA of nonviable cells by using conventional extraction protocols. Furthermore, we provide evidence that prior to denaturing gradient gel electrophoresis, EMA treatment of a mature mixed-population drinking-water biofilm containing a substantial proportion of dead cells can result in community fingerprints dramatically different from those for an untreated biofilm. The interpretation of such fingerprints can have important implications in the field of microbial ecology.Whether bacterial cells are dead or alive is an important question with many implications for monitoring of food and water safety and for analysis of the sterility of pharmaceutical drugs. Due to the relatively long persistence of DNA after cell death, in the range of several days to 3 weeks (10, 14), quantitative analysis of total DNA can lead to a substantial overestimation of the presence of living microorganisms and the accompanying pathogenic threats. The lack of differentiation between DNA from viable and dead bacterial cells is therefore a major obstacle to broad-range application of DNA-based molecular diagnostics (10, 12). The most commonly used strategy to overcome this difficulty is to focus on the presence of the rapidly degrading RNA instead of the stable DNA (16,27,28,31). Due to its rapid turnover, the detection of RNA is far more indicative of the presence of viable cells (1, 2). Nevertheless, working with RNA is technically demanding, and RNA is prone to contamination with RNA-degrading enzymes, resulting in problems of reproducibility. Moreover, the RNA express...

“…Specifically, the phenanthridinium DNA/RNA-intercalating agent enters only those bacteria that have compromised cell walls and membranes and subsequently covalently links to the DNA within the cells (2,4,9,31). Photolysis of EMA by visible light produces a nitrene that covalently links to genomic DNA, cleaving it into small pieces upon photoactivation (9,24,26). Contrastingly, unbound EMA, which remains free in solution, is simultaneously inactivated by reaction with water molecules and no longer capable of covalently binding to DNA (4,10).…”

mentioning

confidence: 99%

Specific Detection of Viable Legionella Cells by Combined Use of Photoactivated Ethidium Monoazide and PCR/Real-Time PCR

Chang

Sugiyama

Taguri³

et al. 2009

Legionella organisms are prevalent in manmade water systems and cause legionellosis in humans. A rapid detection method for viable Legionella cells combining ethidium monoazide (EMA) and PCR/real-time PCR was assessed. EMA could specifically intercalate and cleave the genomic DNA of heat-and chlorine-treated dead Legionella cells. The EMA-PCR assay clearly showed an amplified fragment specific for Legionella DNA from viable cells, but it could not do so for DNA from dead cells. The number of EMA-treated dead Legionella cells estimated by real-time PCR exhibited a 10 4 -to 10 5 -fold decrease compared to the number of dead Legionella cells without EMA treatment. Conversely, no significant difference in the numbers of EMA-treated and untreated viable Legionella cells was detected by the real-time PCR assay. The combined assay was also confirmed to be useful for specific detection of culturable Legionella cells from water samples obtained from spas. Therefore, the combined use of EMA and PCR/real-time PCR detects viable Legionella cells rapidly and specifically and may be useful in environmental surveillance for Legionella.