Determination of viable wine yeast using DNA binding dyes and quantitative PCR

Viable quantitative PCR for assessing the response of Candida albicans to antifungal treatment

“…However, due to the fact that not significant differences were observed between both dyes when viable cells were treated, and since the ∆Cp between live and dead cells were slightly higher (12 Cp of difference) with EMA than with PMA (10 Cp of difference); therefore, EMA was chosen to be more suitable for further viable-qPCR experiments. Similar findings have been reported by Andorrà et al (2010) in wine yeast. These authors showed differences between the mean Cp values from dead and viable cells of around 5 and 12 Cp for EMA and 6-11…”

“…Selective nucleic acid intercalating dyes, such as EMA and PMA, represent one of the most successful recent approaches to detect viable cells (as defined by an intact cell membrane) by PCR (Fittipaldi et al 2012) and have been effectively evaluated in different microorganisms Rudi et al 2005;van Frankenhuyzen et al 2011). As both dyes have similar structures, comparable results might be expected; however, some studies have showed differences (Andorrà et al 2010;Cawthorn et al 2008;Chang et al 2010;Nam et al 2011;Nocker and Camper 2009;Pan and Breidt 2007;Wagner et al 2008). These studies showed that different parameters, such as the dye concentration and the microorganisms analyzed, play an important role in the specificity of both dyes for live and dead cells.…”

“…EMA was demonstrated that penetrates the membranes of live cells, and the extent of EMA entry into intact cells is species-dependent . Thus, multiple optimizations for viable-qPCR techniques have been reported focusing on EMA concentration, incubation time, and photoactivation time (Andorrà et al 2010;Flekna et al 2007;Minami et al 2010;Nogva et al 2003;Pan and Breidt 2007;Wang et al 2009). On the other hand, PMA has been proposed as a more appropriate alternative due to a comparative study showing that PMA is efficiently excluded from cells with intact cell membranes.…”

“…The DNA of dead cells is expected to remain stable over prolonged periods of time (Josephson et al 1993); thus, DNA techniques may include the dead population and overestimate the microorganisms present in a sample. To overcome this, selective nucleic acid intercalating dyes, including ethidium monoazide (EMA) and propidium monoazide 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 5 (PMA), have been successfully used in conjunction with qPCR (herein called viableqPCR) for a large spectrum of microorganisms: bacteria (Agustí et al 2010;Chang et al 2009;Elizaquível et al 2012;Kralik et al 2010;Kramer et al 2009;Takahashi et al 2011), fungi (Andorrà et al 2010;Rawsthorne and Phister 2009;Vesper et al 2008), protozoans (Brescia et al 2009;Fittipaldi et al 2011;Thomas et al 2012), and viruses Parshionikar et al 2010;Sánchez et al 2012). …”

“…As mentioned above, different studies have shown the feasibility of viable-qPCR procedures; nevertheless, there are only four studies in fungi, which are not related with clinical applications (Andorrà et al 2010;Rawsthorne and Phister 2009;Shi et al 2012;Vesper et al 2008).…”