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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...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2015
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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. |
format | Online Article Text |
id | pubmed-6272222 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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