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Two Methods for Increased Specificity and Sensitivity in Loop-Mediated Isothermal Amplification

The technique of loop-mediated isothermal amplification (LAMP) utilizes four (or six) primers targeting six (or eight) regions within a fairly small segment of a genome for amplification, with concentration higher than that used in traditional PCR methods. The high concentrations of primers used lea...

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Autores principales: Wang, De-Guo, Brewster, Jeffrey D., Paul, Moushumi, Tomasula, Peggy M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6272222/
https://www.ncbi.nlm.nih.gov/pubmed/25853320
http://dx.doi.org/10.3390/molecules20046048
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author Wang, De-Guo
Brewster, Jeffrey D.
Paul, Moushumi
Tomasula, Peggy M.
author_facet Wang, De-Guo
Brewster, Jeffrey D.
Paul, Moushumi
Tomasula, Peggy M.
author_sort Wang, De-Guo
collection PubMed
description The technique of loop-mediated isothermal amplification (LAMP) utilizes four (or six) primers targeting six (or eight) regions within a fairly small segment of a genome for amplification, with concentration higher than that used in traditional PCR methods. The high concentrations of primers used leads to an increased likelihood of non-specific amplification induced by primer dimers. In this study, a set of LAMP primers were designed targeting the prfA gene sequence of Listeria monocytogenes, and dimethyl sulfoxide (DMSO) as well as Touchdown LAMP were employed to increase the sensitivity and specificity of the LAMP reactions. The results indicate that the detection limit of this novel LAMP assay with the newly designed primers and additives was 10 fg per reaction, which is ten-fold more sensitive than a commercial Isothermal Amplification Kit and hundred-fold more sensitive than previously reported LAMP assays. This highly sensitive LAMP assay has been shown to detect 11 strains of Listeria monocytogenes, and does not detect other Listeria species (including Listeria innocua and Listeria invanovii), providing some advantages in specificity over commercial Isothermal Amplification Kits and previously reported LAMP assay.
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spelling pubmed-62722222018-12-03 Two Methods for Increased Specificity and Sensitivity in Loop-Mediated Isothermal Amplification Wang, De-Guo Brewster, Jeffrey D. Paul, Moushumi Tomasula, Peggy M. Molecules Article The technique of loop-mediated isothermal amplification (LAMP) utilizes four (or six) primers targeting six (or eight) regions within a fairly small segment of a genome for amplification, with concentration higher than that used in traditional PCR methods. The high concentrations of primers used leads to an increased likelihood of non-specific amplification induced by primer dimers. In this study, a set of LAMP primers were designed targeting the prfA gene sequence of Listeria monocytogenes, and dimethyl sulfoxide (DMSO) as well as Touchdown LAMP were employed to increase the sensitivity and specificity of the LAMP reactions. The results indicate that the detection limit of this novel LAMP assay with the newly designed primers and additives was 10 fg per reaction, which is ten-fold more sensitive than a commercial Isothermal Amplification Kit and hundred-fold more sensitive than previously reported LAMP assays. This highly sensitive LAMP assay has been shown to detect 11 strains of Listeria monocytogenes, and does not detect other Listeria species (including Listeria innocua and Listeria invanovii), providing some advantages in specificity over commercial Isothermal Amplification Kits and previously reported LAMP assay. MDPI 2015-04-07 /pmc/articles/PMC6272222/ /pubmed/25853320 http://dx.doi.org/10.3390/molecules20046048 Text en © 2015 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Wang, De-Guo
Brewster, Jeffrey D.
Paul, Moushumi
Tomasula, Peggy M.
Two Methods for Increased Specificity and Sensitivity in Loop-Mediated Isothermal Amplification
title Two Methods for Increased Specificity and Sensitivity in Loop-Mediated Isothermal Amplification
title_full Two Methods for Increased Specificity and Sensitivity in Loop-Mediated Isothermal Amplification
title_fullStr Two Methods for Increased Specificity and Sensitivity in Loop-Mediated Isothermal Amplification
title_full_unstemmed Two Methods for Increased Specificity and Sensitivity in Loop-Mediated Isothermal Amplification
title_short Two Methods for Increased Specificity and Sensitivity in Loop-Mediated Isothermal Amplification
title_sort two methods for increased specificity and sensitivity in loop-mediated isothermal amplification
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6272222/
https://www.ncbi.nlm.nih.gov/pubmed/25853320
http://dx.doi.org/10.3390/molecules20046048
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