Establishment of a viable cell detection system for microorganisms in wine based on ethidium monoazide and quantitative PCR

Abstract

a b s t r a c tFermentability and contamination level of wine can be assessed through the detection of viable fermentation-related and spoilage-related microorganisms. Ethidium monoazide in combination with quantitative PCR (EMA-qPCR) has been considered as a promising method to enumerate viable cells. Milling for 80 s by Ø 500-mm glass beads is demonstrated to be optimal for DNA extraction from yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) in wine to be used as a template for PCR. EMAqPCR results from experiments using DNA extracted by this method correlate well with the results of a plating assay (R 2 > 0.99), and a PCR efficiency between 96% and 105% was obtained. Moreover, for all of these microorganisms, EMA treatment of pure cultures at a low concentration (10 mg/mL) for 20 min photoactivation resulted in effective differentiation between viable and non-viable cells and had no effect on viable cells. Due to sublethal injury to some cells, underestimation of cell counts was found in most of the wine samples tested using the EMA-qPCR method, and a 40-min incubation in recovery medium could completely offset this error. Our results suggest an optimal glass-bead DNA extraction method and EMA treatment suitable for all of the main microorganisms in wine. The EMA-qPCR method was successfully applied to quantify yeasts, Saccharomyces cerevisiae (S. cerevisiae), LAB, non-Oenococcus oeni LAB (non-O. oeni LAB) and AAB in wine samples.

Molecular Methods in Food Safety Microbiology: Interpretation and Implications of Nucleic Acid Detection

“…Viable/dead stains such as ethidium monoazide or propidium monoazide (EMA or PMA) can be applied prior to PCR to exclude free nucleic acids and those present in damaged bacterial cells or viral capsids from PCR detection, since cell membrane integrity is essential for bacterial viability and an intact virus capsid for virus infectivity (Figure , Box 1, and 3). EMA‐ and PMA‐PCR have been widely and successfully applied to detect or quantify viable microorganisms, including bacteria in the vegetative and spore form, yeasts, and fungi (Vesper and others ; Rawsthorne and Phister ; Rawsthorne and others ; Taskin and others ; Elizaquivel and others ; Radulovic and others ; Shi and others ; Zhu and others ; Blooi and others ; Crespo‐Sempere and others ; Dinu and Bach ; Gensberger and others ; Schnetzinger and others ; Singh and others ). However, EMA and PMA can also diffuse into living bacterial cells with intact membranes under certain conditions, albeit at lower efficiency (Nocker and others ).…”

Progress in understanding preferential detection of live cells using viability dyes in combination with DNA amplification

“…EMA and PMA have since their invention been applied to a wide variety of microorganisms including bacterial vegetative cells (Agusti et al, 2010;Bae and Wuertz, 2009;Cawthorn and Witthuhn, 2008;Delgado Viscogliosi et al, 2009;Pan and Breidt, 2007;Rudi et al, 2005a;Soejima et al, 2007), bacterial spores (Rawsthorne et al, 2009), fungi (Vesper et al, 2008), viruses Graiver et al, 2010;Sanchez et al, 2012), yeast (Andorrà et al, 2010;Shi et al, 2012), and protozoa (Brescia et al, 2009;Fittipaldi et al, 2011a). The addition of a pre-treatment step to the sample analysis to inhibit the amplification of DNA from membrane-damaged cells has been used in combination with end point PCR (Brescia et al, 2009), real-time or quantitative PCR (qPCR) (Rudi et al, 2005a), reverse transcription PCR (Graiver et al, 2010), isothermal amplification Lu et al, 2009;Wang et al, 2012), denaturing gradient gel electrophoresis (DGGE) (Nocker et al, 2007a), terminal restriction fragment length polymorphism (TRFLP) (Rogers et al, 2008), microarray technology , and nextgeneration sequencing .…”

Development of loop-mediated isothermal amplification assays for genotyping of Type III Secretion System in Pseudomonas aeruginosa

“…Glass‐bead DNA extraction, DNA purification and DNA qualification were performed according to Shi et al . (2012b). For sensitivity tests, DNA was diluted in ddH 2 O to concentration of 10 pg, 100 fg and 10 fg per reaction.…”

“…Five hundred microliters of P. aeruginosa cultures were centrifuged at 12 000 g for 5 min and the supernatant was removed. Glass-bead DNA extraction, DNA purification and DNA qualification were performed according to Shi et al (2012b). For sensitivity tests, DNA was diluted in ddH 2 O to concentration of 10 pg, 100 fg and 10 fg per reaction.…”