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Real-time quantitative PCR to discriminate and quantify lambdoid bacteriophages of Escherichia coli K-12

Quantification of bacteriophages by real-time quantitative PCR (qPCR) is an interesting alternative to the traditional plaque assay. Importantly, the method should in principle be able to discriminate between closely related phages that are indistinguishable by most other means. Here, a method is pr...

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Detalles Bibliográficos
Autor principal: Refardt, Dominik
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Landes Bioscience 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3442831/
https://www.ncbi.nlm.nih.gov/pubmed/23050220
http://dx.doi.org/10.4161/bact.20092
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author Refardt, Dominik
author_facet Refardt, Dominik
author_sort Refardt, Dominik
collection PubMed
description Quantification of bacteriophages by real-time quantitative PCR (qPCR) is an interesting alternative to the traditional plaque assay. Importantly, the method should in principle be able to discriminate between closely related phages that are indistinguishable by most other means. Here, a method is presented that employs qPCR to discriminate and quantify ten closely related lambdoid phages of Escherichia coli str. K-12. It is shown that (1) treatment of samples with DNase efficiently removes non-encapsidated DNA, while the titer of plaque forming units is not affected, (2) individual phage types can be accurately quantified in mixed lysates, and (3) the detection limit corresponds to that of a plaque assay. The method is used to quantify individual phage types that are released from lysogens that carry up to three different prophages.
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spelling pubmed-34428312012-10-05 Real-time quantitative PCR to discriminate and quantify lambdoid bacteriophages of Escherichia coli K-12 Refardt, Dominik Bacteriophage Methods and Protocols Quantification of bacteriophages by real-time quantitative PCR (qPCR) is an interesting alternative to the traditional plaque assay. Importantly, the method should in principle be able to discriminate between closely related phages that are indistinguishable by most other means. Here, a method is presented that employs qPCR to discriminate and quantify ten closely related lambdoid phages of Escherichia coli str. K-12. It is shown that (1) treatment of samples with DNase efficiently removes non-encapsidated DNA, while the titer of plaque forming units is not affected, (2) individual phage types can be accurately quantified in mixed lysates, and (3) the detection limit corresponds to that of a plaque assay. The method is used to quantify individual phage types that are released from lysogens that carry up to three different prophages. Landes Bioscience 2012-04-01 /pmc/articles/PMC3442831/ /pubmed/23050220 http://dx.doi.org/10.4161/bact.20092 Text en Copyright © 2012 Landes Bioscience http://creativecommons.org/licenses/by-nc/3.0/ This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.
spellingShingle Methods and Protocols
Refardt, Dominik
Real-time quantitative PCR to discriminate and quantify lambdoid bacteriophages of Escherichia coli K-12
title Real-time quantitative PCR to discriminate and quantify lambdoid bacteriophages of Escherichia coli K-12
title_full Real-time quantitative PCR to discriminate and quantify lambdoid bacteriophages of Escherichia coli K-12
title_fullStr Real-time quantitative PCR to discriminate and quantify lambdoid bacteriophages of Escherichia coli K-12
title_full_unstemmed Real-time quantitative PCR to discriminate and quantify lambdoid bacteriophages of Escherichia coli K-12
title_short Real-time quantitative PCR to discriminate and quantify lambdoid bacteriophages of Escherichia coli K-12
title_sort real-time quantitative pcr to discriminate and quantify lambdoid bacteriophages of escherichia coli k-12
topic Methods and Protocols
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3442831/
https://www.ncbi.nlm.nih.gov/pubmed/23050220
http://dx.doi.org/10.4161/bact.20092
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