Improving efficiency of viability-PCR for selective detection of live cells

Abstract

Viability PCR (v-PCR) as a method to selectively detect intact live cells has gained considerable interest over the last years with an increasing number of applications. The principle is based on treatment of microbiological samples with a viability dye prior to extraction of genomic DNA and its amplification. The dye is selectively taken up by membrane-compromised dead cells resulting in the degradation of their DNA upon light exposure and therefore inhibition of amplification. Although the treatment greatly helps to generate more meaningful data, one of the main drawbacks of the technique is currently that the exclusion of dead cell signals can be incomplete leading to false-positive signals. The resulting overestimation of live cell population is especially problematic for the detection of pathogens. We assessed in this study different conditions to increase the penetration of propidium monoazide (PMA) into dead cells of Salmonella Typhimurium and Listeria monocytogenes as representatives of gram-negative and gram-positive bacteria. When working with a low dye concentration of 10μM, a strong relationship of PMA treatment efficiency with temperature and incubation time was observed. Exposing cells to PMA at a temperature exceeding the growth temperature by 10°C for 30min proved greatly beneficial. Co-incubation of cells with PMA and deoxycholate on the other hand was only beneficial for Salmonella, but resulted in a strong undesired uptake of PMA by live Listeria cells. This difference is in agreement with the gram-specific effect of the bile salt during growth.