Differentiation of genes extracted from non-viable versus viable micro-organisms in environmental samples using...

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.