Development and validation of qualitative SYBR®Green Real-Time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes

A combination of four qualitative SYBR®Green qPCR screening assays targeting two levels of discrimination: Listeria genus (except Listeria grayi) and Listeria monocytogenes, is presented. These assays have been developed to be run simultaneously using the same polymerase chain reaction (PCR) program...

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Autores principales: Barbau-Piednoir, Elodie, Botteldoorn, Nadine, Yde, Marc, Mahillon, Jacques, Roosens, Nancy H.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer-Verlag 2012
Materias:
Applied Genetics and Molecular Biotechnology
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3627020/
https://www.ncbi.nlm.nih.gov/pubmed/23086339
http://dx.doi.org/10.1007/s00253-012-4477-2
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author Barbau-Piednoir, Elodie
Botteldoorn, Nadine
Yde, Marc
Mahillon, Jacques
Roosens, Nancy H.
author_facet Barbau-Piednoir, Elodie
Botteldoorn, Nadine
Yde, Marc
Mahillon, Jacques
Roosens, Nancy H.
author_sort Barbau-Piednoir, Elodie
collection PubMed
description A combination of four qualitative SYBR®Green qPCR screening assays targeting two levels of discrimination: Listeria genus (except Listeria grayi) and Listeria monocytogenes, is presented. These assays have been developed to be run simultaneously using the same polymerase chain reaction (PCR) programme. The paper also proposes a new validation procedure to specifically validate qPCR assays applied to food microbiology according to two guidelines: the ISO 22118 norm and the “Definition of minimum performance requirements for analytical methods of GMO testing”. The developed assays target the iap, prs and hlyA genes that belong to or neighbour the virulence cluster of Listeria spp. The selected primers were designed to amplify short fragments (60 to 103 bp) in order to obtain optimal PCR efficiency (between 97 and 107 % efficiency). The limit of detection of the SYBR®Green qPCR assays is two to five copies of target genes per qPCR reaction. These assays are highly accurate (98.08 and 100 % accuracy for the Listeria spp. and L. monocytogenes assays, respectively).
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spelling pubmed-36270202013-04-17 Development and validation of qualitative SYBR®Green Real-Time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes Barbau-Piednoir, Elodie Botteldoorn, Nadine Yde, Marc Mahillon, Jacques Roosens, Nancy H. Appl Microbiol Biotechnol Applied Genetics and Molecular Biotechnology A combination of four qualitative SYBR®Green qPCR screening assays targeting two levels of discrimination: Listeria genus (except Listeria grayi) and Listeria monocytogenes, is presented. These assays have been developed to be run simultaneously using the same polymerase chain reaction (PCR) programme. The paper also proposes a new validation procedure to specifically validate qPCR assays applied to food microbiology according to two guidelines: the ISO 22118 norm and the “Definition of minimum performance requirements for analytical methods of GMO testing”. The developed assays target the iap, prs and hlyA genes that belong to or neighbour the virulence cluster of Listeria spp. The selected primers were designed to amplify short fragments (60 to 103 bp) in order to obtain optimal PCR efficiency (between 97 and 107 % efficiency). The limit of detection of the SYBR®Green qPCR assays is two to five copies of target genes per qPCR reaction. These assays are highly accurate (98.08 and 100 % accuracy for the Listeria spp. and L. monocytogenes assays, respectively). Springer-Verlag 2012-10-20 2013 /pmc/articles/PMC3627020/ /pubmed/23086339 http://dx.doi.org/10.1007/s00253-012-4477-2 Text en © The Author(s) 2012 https://creativecommons.org/licenses/by/4.0/ This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
spellingShingle Applied Genetics and Molecular Biotechnology
Barbau-Piednoir, Elodie
Botteldoorn, Nadine
Yde, Marc
Mahillon, Jacques
Roosens, Nancy H.
Development and validation of qualitative SYBR®Green Real-Time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes
title Development and validation of qualitative SYBR®Green Real-Time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes
title_full Development and validation of qualitative SYBR®Green Real-Time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes
title_fullStr Development and validation of qualitative SYBR®Green Real-Time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes
title_full_unstemmed Development and validation of qualitative SYBR®Green Real-Time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes
title_short Development and validation of qualitative SYBR®Green Real-Time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes
title_sort development and validation of qualitative sybr®green real-time pcr for detection and discrimination of listeria spp. and listeria monocytogenes
topic Applied Genetics and Molecular Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3627020/
https://www.ncbi.nlm.nih.gov/pubmed/23086339
http://dx.doi.org/10.1007/s00253-012-4477-2
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