Differentiation of genes extracted from non-viable versus viable micro-organisms in environmental samples using ethidium monoazide bromide Jola M. Pisz a , John R. Lawrence b , Alexis N. Schafer a,c , Steven D. Siciliano a,c, ⁎ a Department of Soil Science, 51 Campus Drive, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5A8 b National Water Research Institute, 11 Innovation Boulevard, Saskatoon, Saskatchewan, Canada S7N 3H5 c Toxicology Group, University of Saskatchewan, Saskatoon, Saskatchewan, Canada Received 4 September 2007; received in revised form 20 September 2007; accepted 20 September 2007 Available online 25 October 2007 Abstract Differentiation of DNA derived from viable or non-viable microorganisms within mixed microbial communities continues to be one of the greatest challenges in molecular studies of environmental samples. A novel method developed for microbial food pathogens is tested here on environmental samples. This technique involves the use of ethidium monoazide bromide (EMA) for the distinction of live/dead cells. In non- viable cells EMA intercalates into the DNA which prevents amplification by PCR. We adapted and evaluated the EMA technique for soil, elemental sulfur and river biofilm samples. Quantitative PCR determined that EMA suppressed 99.99% of E. coli LKI gfp+ signal in non-viable cultures and 100.00% when the cultures were added to soil samples. The same technique was also successful at suppressing DNA amplification from spiked non-viable cells in elemental sulfur samples by 100.00%, but not in three Saskatchewan River biofilms. In sub Antarctic soil, EMA- Q-PCR was used to detect the prevalence of a functional gene, amoA, and this was closely correlated to nitrification activity measurements. The ability of EMA to differentiate between viable and non-viable populations in soil was confirmed by the similarity of the 16S rRNA denaturing- gradient-gel electrophoresis DNA fingerprint of EMA treated soil and the 16S rRNA cDNA fingerprint of non-EMA treated soil. The EMA technique effectively suppressed amplification of non-viable spiked controls, closely mirrored activity assays and yielded community composition profiles similar to rRNA techniques. The use of EMA in soil effectively suppressed amplification of non-viable organism DNA, however it was not effective in biofilm samples and EMA partially inhibited amplification of viable organism DNA in elemental sulfur samples. © 2007 Elsevier B.V. All rights reserved. Keywords: Ethidium monoazide bromide; Quantitative PCR; Environmental samples; Elemental sulfur; Biofilm; Soil 1. Introduction Molecular analysis of bacterial communities in the environ- ment is an important tool for the advancement of modern microbial ecology. However, it is often limited by the inability to differentiate between genes extracted from non-viable versus viable organisms. Many techniques have been developed for monocultural applications, such as flow cytometry, single cell sorting, and autoradiography, but are rarely successful in mixed microbial populations (O'Brien and Bolton, 1995; Rudi et al., 2005a). For functional genes, such as ammonia mono-oxygenase, current methods, such as mRNA extractions, are technically challenging. Recently an intercalating ethidium bromide deriva- tive has gained attention for use in quantitative gene assays (Lee and Levin, 2006; Nogva et al., 2003; Rudi et al., 2005a,b; Wang and Levin, 2006). Ethidium monoazide bromide (EMA) (Invitrogen, Burling- ton, ON) is unable to enter cells with intact membranes, and so it can only attach to free DNA or DNA that is present within cells with compromised membranes. Upon exposure to high intensity visible light, EMA produces a highly reactive nitrene which covalently binds to DNA. Unbound EMA converts to a hydroxylamine derivative, rendering it incapable of further Available online at www.sciencedirect.com Journal of Microbiological Methods 71 (2007) 312 – 318 www.elsevier.com/locate/jmicmeth ⁎ Corresponding author. Department of Soil Science, 51 Campus Drive, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5A8. Tel.: +1 306 966 4035; fax: +1 306 966 6881. E-mail address: steven.siciliano@usask.ca (S.D. Siciliano). 0167-7012/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2007.09.015