Viable real-time PCR in environmental samples: can all data be interpreted directly?

Microbial Ecology
Mariana FittipaldiJordi Morató

Abstract

Selective nucleic acid intercalating dyes--ethidium monoazide (EMA) and propidium monoazide (PMA)--represent one of the most successful recent approaches to detect viable cells (as defined by an intact cell membrane) by PCR and have been effectively evaluated in different microorganisms. However, some practical limitations were found, especially in environmental samples. The aim of this work was to show that in the application of viable real-time PCR, there may be significant biases and to propose a strategy for overcoming some of these problems. We present an approach based on the combination of three real-time PCR amplifications for each sample that should provide an improved estimation of the number of viable cells. This approach could be useful especially when it is difficult to determine a priori how to optimize methods using PMA or EMA. Although further studies are required to improve viable real-time PCR methods, the concept as outlined here presents an interesting future research direction.

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Citations

Oct 12, 2012·The ISME Journal·Parag VaishampayanKasthuri Venkateswaran
Mar 4, 2014·Critical Reviews in Microbiology·Harriet Whiley, Michael Taylor
Jul 22, 2014·International Journal of Environmental Research and Public Health·Harriet WhileyRichard Bentham
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Sep 4, 2012·Journal of Microbiological Methods·Mariana FittipaldiFrancesc Codony
Sep 23, 2014·Molecular and Cellular Probes·Savina DitommasoCarla M Zotti
May 3, 2016·Diagnostic Microbiology and Infectious Disease·Maria ScaturroMaria Luisa Ricci
Feb 28, 2017·PLoS Neglected Tropical Diseases·Roman ThibeauxCyrille Goarant
Mar 13, 2016·Applied and Environmental Microbiology·Isabella MarchesiAnnalisa Bargellini
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Dec 7, 2016·Frontiers in Microbiology·Dexin ZengBaoguang Li
Oct 16, 2020·Letters in Applied Microbiology·S CopinM Bonnin-Jusserand

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