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Quantification of viable Legionella pneumophila cells using propidium monoazide combined with quantitative PCR.
MedLine Citation:
PMID:  21329735     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
One of the greatest challenges of implementing fast molecular detection methods as part of Legionella surveillance systems is to limit detection to live cells. In this work, a protocol for sample treatment with propidium monoazide (PMA) in combination with quantitative PCR (qPCR) has been optimized and validated for L. pneumophila as an alternative of the currently used time-consuming culture method. Results from PMA-qPCR were compared with culture isolation and traditional qPCR. Under the conditions used, sample treatment with 50μM PMA followed by 5min of light exposure were assumed optimal resulting in an average reduction of 4.45 log units of the qPCR signal from heat-killed cells. When applied to environmental samples (including water from cooling water towers, hospitals, spas, hot water systems in hotels, and tap water), different degrees of correlations between the three methods were obtained which might be explained by different matrix properties, but also varying degrees of non-culturable cells. It was furthermore shown that PMA displayed substantially lower cytotoxicity with Legionella than the alternative dye ethidium monoazide (EMA) when exposing live cells to the dye followed by plate counting. This result confirmed findings with other species that PMA is less membrane-permeant and more selective for intact cells. In conclusion, PMA-qPCR is a promising technique for limiting detection to intact cells and makes Legionella surveillance data substantially more relevant in comparison with qPCR alone. For future research it would be desirable to increase the method's capacity to exclude signals from dead cells in difficult matrices or samples containing high numbers of dead cells.
Authors:
M Adela Yáñez; Andreas Nocker; Elena Soria-Soria; Raquel Múrtula; Lorena Martínez; Vicente Catalán
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2011-2-14
Journal Detail:
Title:  Journal of microbiological methods     Volume:  -     ISSN:  1872-8359     ISO Abbreviation:  -     Publication Date:  2011 Feb 
Date Detail:
Created Date:  2011-2-18     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8306883     Medline TA:  J Microbiol Methods     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
Copyright © 2010. Published by Elsevier B.V.
Affiliation:
LABAQUA, S.A., c) Del Dracma, 16-18, 0314 Alicante Spain.
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