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Development of a visual multiplex fluorescent LAMP assay for the detection of foot-and-mouth disease, vesicular stomatitis and bluetongue viruses

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that can be used to amplify target genes at a constant temperature, and it has several advantages, including convenience, specificity and sensitivity. However, due to the special interpretation methods of this te...

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Autores principales: Fan, Qing, Xie, Zhixun, Wei, You, Zhang, Yanfang, Xie, Zhiqin, Xie, Liji, Huang, Jiaoling, Zeng, Tingting, Wang, Sheng, Luo, Sisi, Li, Meng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9731490/
https://www.ncbi.nlm.nih.gov/pubmed/36480573
http://dx.doi.org/10.1371/journal.pone.0278451
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author Fan, Qing
Xie, Zhixun
Wei, You
Zhang, Yanfang
Xie, Zhiqin
Xie, Liji
Huang, Jiaoling
Zeng, Tingting
Wang, Sheng
Luo, Sisi
Li, Meng
author_facet Fan, Qing
Xie, Zhixun
Wei, You
Zhang, Yanfang
Xie, Zhiqin
Xie, Liji
Huang, Jiaoling
Zeng, Tingting
Wang, Sheng
Luo, Sisi
Li, Meng
author_sort Fan, Qing
collection PubMed
description Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that can be used to amplify target genes at a constant temperature, and it has several advantages, including convenience, specificity and sensitivity. However, due to the special interpretation methods of this technology for reaction results, all the previously reported LAMP detection methods have been restricted to identifying a single target, which limits the application of this technology. In this study, we modified conventional LAMP to include a quencher-fluorophore composite probe complementary to the F1c segment of the inner primer FIP; upon strand separation, a gain in the visible fluorescent signal was observed. The probes could be labeled with different fluorophores, showing different colors at the corresponding wavelengths. Therefore, this multiplex LAMP (mLAMP) assay can simultaneously detect 1–3 target sequences in a single LAMP reaction tube, and the results are more accurate and intuitive. In this study, we comprehensively demonstrated a single-reaction mLAMP assay for the robust detection of three cattle viruses without nonspecific amplification of other related pathogenic cattle viruses. The detection limit of this mLAMP assay was as low as 526–2477 copies/reaction for the recombinant plasmids. It is expected that this mLAMP assay can be widely used in clinical diagnosis.
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spelling pubmed-97314902022-12-09 Development of a visual multiplex fluorescent LAMP assay for the detection of foot-and-mouth disease, vesicular stomatitis and bluetongue viruses Fan, Qing Xie, Zhixun Wei, You Zhang, Yanfang Xie, Zhiqin Xie, Liji Huang, Jiaoling Zeng, Tingting Wang, Sheng Luo, Sisi Li, Meng PLoS One Research Article Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that can be used to amplify target genes at a constant temperature, and it has several advantages, including convenience, specificity and sensitivity. However, due to the special interpretation methods of this technology for reaction results, all the previously reported LAMP detection methods have been restricted to identifying a single target, which limits the application of this technology. In this study, we modified conventional LAMP to include a quencher-fluorophore composite probe complementary to the F1c segment of the inner primer FIP; upon strand separation, a gain in the visible fluorescent signal was observed. The probes could be labeled with different fluorophores, showing different colors at the corresponding wavelengths. Therefore, this multiplex LAMP (mLAMP) assay can simultaneously detect 1–3 target sequences in a single LAMP reaction tube, and the results are more accurate and intuitive. In this study, we comprehensively demonstrated a single-reaction mLAMP assay for the robust detection of three cattle viruses without nonspecific amplification of other related pathogenic cattle viruses. The detection limit of this mLAMP assay was as low as 526–2477 copies/reaction for the recombinant plasmids. It is expected that this mLAMP assay can be widely used in clinical diagnosis. Public Library of Science 2022-12-08 /pmc/articles/PMC9731490/ /pubmed/36480573 http://dx.doi.org/10.1371/journal.pone.0278451 Text en © 2022 Fan et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Fan, Qing
Xie, Zhixun
Wei, You
Zhang, Yanfang
Xie, Zhiqin
Xie, Liji
Huang, Jiaoling
Zeng, Tingting
Wang, Sheng
Luo, Sisi
Li, Meng
Development of a visual multiplex fluorescent LAMP assay for the detection of foot-and-mouth disease, vesicular stomatitis and bluetongue viruses
title Development of a visual multiplex fluorescent LAMP assay for the detection of foot-and-mouth disease, vesicular stomatitis and bluetongue viruses
title_full Development of a visual multiplex fluorescent LAMP assay for the detection of foot-and-mouth disease, vesicular stomatitis and bluetongue viruses
title_fullStr Development of a visual multiplex fluorescent LAMP assay for the detection of foot-and-mouth disease, vesicular stomatitis and bluetongue viruses
title_full_unstemmed Development of a visual multiplex fluorescent LAMP assay for the detection of foot-and-mouth disease, vesicular stomatitis and bluetongue viruses
title_short Development of a visual multiplex fluorescent LAMP assay for the detection of foot-and-mouth disease, vesicular stomatitis and bluetongue viruses
title_sort development of a visual multiplex fluorescent lamp assay for the detection of foot-and-mouth disease, vesicular stomatitis and bluetongue viruses
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9731490/
https://www.ncbi.nlm.nih.gov/pubmed/36480573
http://dx.doi.org/10.1371/journal.pone.0278451
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