Ethidium Monoazide for DNA-Based Differentiation of Viable and Dead Bacteria by 5′-Nuclease PCR

Abstract

PCR techniques have significantly improved the detection and identification of bacterial pathogens. Even so, the lack of differentiation between DNA from viable and dead cells is one of the major challenges for diagnostic DNA-based methods. Certain nucleic acid-binding dyes can selectively enter dead bacteria and subsequently be covalently linked to DNA. Ethidium monoazide (EMA) is a DNA intercalating dye that enters bacteria with damaged membranes. This dye can be covalently linked to DNA by photoactivation. Our goal was to utilize the irreversible binding of photoactivated EMA to DNA to inhibit the PCR of DNA from dead bacteria. Quantitative 5′-nuclease PCR assays were used to measure the effect of EMA. The conclusion from the experiments was that EMA covalently bound to DNA inhibited the 5′-nuclease PCR. The maximum inhibition of PCR on pure DNA cross-linked with EMA gave a signal reduction of approximately −4.5 log units relative to untreated DNA. The viable/dead differentiation with the EMA method was evaluated through comparison with BacLightTM staining (microscopic examination) and plate counts. The EMA and BacLight methods gave corresponding results for all bacteria and conditions tested. Furthermore, we obtained a high correlation between plate counts and the EMA results for bacteria killed with ethanol, benzalkonium chloride (disinfectant), or exposure to 70°C. However, for bacteria exposed to 100°C, the number of viable cells recovered by plating was lower than the detection limit with the EMA method. In conclusion, the EMA method is promising for DNA-based differentiation between viable and dead bacteria.