Figure 2. Use of EMA-PCR for enumerating living C. burnetii cells.

Ethidium monoazide (EMA)-PCR was used to count live C. burnetii cells. A) Schematic representation of the EMA-PCR principle. EMA is unable to diffuse through intact bacterial cells with a non-damaged cell wall. Therefore, subsequent detection of those cells by qPCR is not hindered (left side). Following diffusion inside permeable cells, EMA creates covalent cross-links in the DNA molecule upon exposure to a blue-light source. DNA originating from those cells will not be amplified by qPCR (right side). B) EMA-PCR on dead C. burnetii bacterial cells. C. burnetii bacterial cells killed by heating at various time points and treated with EMA stain failed to yield positive PCR reactions. C) Repeatability and reproducibility of EMA-PCR measurements. Two operators using the same protocol and the same three stock-aliquots of C. burnetii determined the bacterial load in independent measurements. To assess intra-assays repeatability, five measurements were collected for each time point.