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Helix loop-mediated isothermal amplification of nucleic acids
Isothermal nucleic acid amplification has played a key role in the point of care test (POCT). In this study, a helix loop-mediated isothermal amplification (HAMP) method with high specificity, efficiency and rapidity was developed. The MERS-Cov orf1b gene was chosen for the validation and optimizati...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Royal Society of Chemistry
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9080610/ https://www.ncbi.nlm.nih.gov/pubmed/35539645 http://dx.doi.org/10.1039/c8ra01201f |
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author | Mao, Rui Qi, Lifei Wang, Zhuo Liu, Hongtao Du, Yuguang |
author_facet | Mao, Rui Qi, Lifei Wang, Zhuo Liu, Hongtao Du, Yuguang |
author_sort | Mao, Rui |
collection | PubMed |
description | Isothermal nucleic acid amplification has played a key role in the point of care test (POCT). In this study, a helix loop-mediated isothermal amplification (HAMP) method with high specificity, efficiency and rapidity was developed. The MERS-Cov orf1b gene was chosen for the validation and optimization of HAMP. The HAMP analysis was performed at a constant temperature of 61–65 °C and yielded a self-primed spiral structure with no introduction of exogenous gene sequence by two pairs of specially designed primers. The primers for helix loop formation were composed of two complementary primers including the helix forward primer and the helix reverse primer, the 3′ ends of which were complementary to their respective target nucleic acids. HAMP assay can be monitored by fluorescence signals with the addition of Eva Green in the reaction mixture. In addition, an accelerated HAMP was developed after the addition of acceleration probe, which could be finished within 75 min with a sensitivity of 10 copies per reaction. Further, a reverse transcription-HAMP (RT-HAMP) was proven to be feasible for RNA detection by combining the reverse transcriptase with DNA polymerase. Finally, both the HAMP and RT-HAMP assay were visually conducted by using Hydroxynaphthol blue (HNB) as a chromogenic indicator. All in all, it is suggested that the HAMP assay would have great potential in POCT applications. |
format | Online Article Text |
id | pubmed-9080610 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | The Royal Society of Chemistry |
record_format | MEDLINE/PubMed |
spelling | pubmed-90806102022-05-09 Helix loop-mediated isothermal amplification of nucleic acids Mao, Rui Qi, Lifei Wang, Zhuo Liu, Hongtao Du, Yuguang RSC Adv Chemistry Isothermal nucleic acid amplification has played a key role in the point of care test (POCT). In this study, a helix loop-mediated isothermal amplification (HAMP) method with high specificity, efficiency and rapidity was developed. The MERS-Cov orf1b gene was chosen for the validation and optimization of HAMP. The HAMP analysis was performed at a constant temperature of 61–65 °C and yielded a self-primed spiral structure with no introduction of exogenous gene sequence by two pairs of specially designed primers. The primers for helix loop formation were composed of two complementary primers including the helix forward primer and the helix reverse primer, the 3′ ends of which were complementary to their respective target nucleic acids. HAMP assay can be monitored by fluorescence signals with the addition of Eva Green in the reaction mixture. In addition, an accelerated HAMP was developed after the addition of acceleration probe, which could be finished within 75 min with a sensitivity of 10 copies per reaction. Further, a reverse transcription-HAMP (RT-HAMP) was proven to be feasible for RNA detection by combining the reverse transcriptase with DNA polymerase. Finally, both the HAMP and RT-HAMP assay were visually conducted by using Hydroxynaphthol blue (HNB) as a chromogenic indicator. All in all, it is suggested that the HAMP assay would have great potential in POCT applications. The Royal Society of Chemistry 2018-05-23 /pmc/articles/PMC9080610/ /pubmed/35539645 http://dx.doi.org/10.1039/c8ra01201f Text en This journal is © The Royal Society of Chemistry https://creativecommons.org/licenses/by-nc/3.0/ |
spellingShingle | Chemistry Mao, Rui Qi, Lifei Wang, Zhuo Liu, Hongtao Du, Yuguang Helix loop-mediated isothermal amplification of nucleic acids |
title | Helix loop-mediated isothermal amplification of nucleic acids |
title_full | Helix loop-mediated isothermal amplification of nucleic acids |
title_fullStr | Helix loop-mediated isothermal amplification of nucleic acids |
title_full_unstemmed | Helix loop-mediated isothermal amplification of nucleic acids |
title_short | Helix loop-mediated isothermal amplification of nucleic acids |
title_sort | helix loop-mediated isothermal amplification of nucleic acids |
topic | Chemistry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9080610/ https://www.ncbi.nlm.nih.gov/pubmed/35539645 http://dx.doi.org/10.1039/c8ra01201f |
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