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 le...
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| Format: | Article |
| Language: | English |
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MDPI AG
2015-04-01
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| Series: | Molecules |
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| Online Access: | http://www.mdpi.com/1420-3049/20/4/6048 |
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| author | De-Guo Wang Jeffrey D. Brewster Moushumi Paul Peggy M. Tomasula |
| author_facet | De-Guo Wang Jeffrey D. Brewster Moushumi Paul Peggy M. Tomasula |
| author_sort | De-Guo Wang |
| collection | DOAJ |
| 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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| format | Article |
| id | doaj.art-6b114ef9e7a14c6eb48761cc0135d790 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-12-12T21:04:50Z |
| publishDate | 2015-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-6b114ef9e7a14c6eb48761cc0135d7902022-12-22T00:12:02ZengMDPI AGMolecules1420-30492015-04-012046048605910.3390/molecules20046048molecules20046048Two Methods for Increased Specificity and Sensitivity in Loop-Mediated Isothermal AmplificationDe-Guo Wang0Jeffrey D. Brewster1Moushumi Paul2Peggy M. Tomasula3Henan Postdoctoral Research Base, Food and Bioengineering College, Xuchang University, Xuchang 461000, ChinaEastern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Wyndmoor, PA 19038, USAEastern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Wyndmoor, PA 19038, USAEastern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Wyndmoor, PA 19038, USAThe 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.http://www.mdpi.com/1420-3049/20/4/6048loop-mediated isothermal amplification (LAMP)non-specific amplificationdimethyl sulfoxide (DMSO)Touchdown LAMP |
| spellingShingle | De-Guo Wang Jeffrey D. Brewster Moushumi Paul Peggy M. Tomasula Two Methods for Increased Specificity and Sensitivity in Loop-Mediated Isothermal Amplification Molecules loop-mediated isothermal amplification (LAMP) non-specific amplification dimethyl sulfoxide (DMSO) Touchdown LAMP |
| 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 | loop-mediated isothermal amplification (LAMP) non-specific amplification dimethyl sulfoxide (DMSO) Touchdown LAMP |
| url | http://www.mdpi.com/1420-3049/20/4/6048 |
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