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Comparison of Biosensing Methods Based on Different Isothermal Amplification Strategies: A Case Study with Erwinia amylovora

Isothermal amplifications allow for the highly sensitive detection of nucleic acids, bypassing the use of instrumental thermal cycling. This work aimed to carry out an experimental comparison of the four most promising techniques: recombinase polymerase amplification (RPA) and loop-mediated isotherm...

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Autores principales: Ivanov, Aleksandr V., Safenkova, Irina V., Drenova, Natalia V., Zherdev, Anatoly V., Dzantiev, Boris B.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9776058/
https://www.ncbi.nlm.nih.gov/pubmed/36551141
http://dx.doi.org/10.3390/bios12121174
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author Ivanov, Aleksandr V.
Safenkova, Irina V.
Drenova, Natalia V.
Zherdev, Anatoly V.
Dzantiev, Boris B.
author_facet Ivanov, Aleksandr V.
Safenkova, Irina V.
Drenova, Natalia V.
Zherdev, Anatoly V.
Dzantiev, Boris B.
author_sort Ivanov, Aleksandr V.
collection PubMed
description Isothermal amplifications allow for the highly sensitive detection of nucleic acids, bypassing the use of instrumental thermal cycling. This work aimed to carry out an experimental comparison of the four most promising techniques: recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) coupled with lateral flow test or coupled with additional amplification based on CRISPR/Cas12a resulting from the fluorescence of the Cas12a-cleaved probe. To compare the four amplification techniques, we chose the bacterial phytopathogen Erwinia amylovora (causative agent of fire blight), which has a quarantine significance in many countries and possesses a serious threat to agriculture. Three genes were chosen as the targets and primers were selected for each one (two for RPA and six for LAMP). They were functionalized by labels (biotin, fluorescein) at the 5′ ends for amplicons recognition by LFT. As a result, we developed LAMP-LFT, LAMP-CRISPR/Cas, RPA-LFT, and RPA-CRISPR/Cas for E. amylovora detection. The detection limit was 10(4) CFU/mL for LAMP-LFT, 10(3) CFU/mL for LAMP-CRISPR/Cas, and 10(2) CFU/mL for RPA-LFT and RPA-CRISPR/Cas. The results of four developed test systems were verified by qPCR on a panel of real samples. The developed assays based on RPA, LAMP, CRISPR/Cas12a, and LFT are rapid (30–55 min), user-friendly, and highly sensitive for E. amylovora detection. All proposed detection methods can be applied to fire blight diagnosis and effective management of this disease.
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spelling pubmed-97760582022-12-23 Comparison of Biosensing Methods Based on Different Isothermal Amplification Strategies: A Case Study with Erwinia amylovora Ivanov, Aleksandr V. Safenkova, Irina V. Drenova, Natalia V. Zherdev, Anatoly V. Dzantiev, Boris B. Biosensors (Basel) Article Isothermal amplifications allow for the highly sensitive detection of nucleic acids, bypassing the use of instrumental thermal cycling. This work aimed to carry out an experimental comparison of the four most promising techniques: recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) coupled with lateral flow test or coupled with additional amplification based on CRISPR/Cas12a resulting from the fluorescence of the Cas12a-cleaved probe. To compare the four amplification techniques, we chose the bacterial phytopathogen Erwinia amylovora (causative agent of fire blight), which has a quarantine significance in many countries and possesses a serious threat to agriculture. Three genes were chosen as the targets and primers were selected for each one (two for RPA and six for LAMP). They were functionalized by labels (biotin, fluorescein) at the 5′ ends for amplicons recognition by LFT. As a result, we developed LAMP-LFT, LAMP-CRISPR/Cas, RPA-LFT, and RPA-CRISPR/Cas for E. amylovora detection. The detection limit was 10(4) CFU/mL for LAMP-LFT, 10(3) CFU/mL for LAMP-CRISPR/Cas, and 10(2) CFU/mL for RPA-LFT and RPA-CRISPR/Cas. The results of four developed test systems were verified by qPCR on a panel of real samples. The developed assays based on RPA, LAMP, CRISPR/Cas12a, and LFT are rapid (30–55 min), user-friendly, and highly sensitive for E. amylovora detection. All proposed detection methods can be applied to fire blight diagnosis and effective management of this disease. MDPI 2022-12-15 /pmc/articles/PMC9776058/ /pubmed/36551141 http://dx.doi.org/10.3390/bios12121174 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Ivanov, Aleksandr V.
Safenkova, Irina V.
Drenova, Natalia V.
Zherdev, Anatoly V.
Dzantiev, Boris B.
Comparison of Biosensing Methods Based on Different Isothermal Amplification Strategies: A Case Study with Erwinia amylovora
title Comparison of Biosensing Methods Based on Different Isothermal Amplification Strategies: A Case Study with Erwinia amylovora
title_full Comparison of Biosensing Methods Based on Different Isothermal Amplification Strategies: A Case Study with Erwinia amylovora
title_fullStr Comparison of Biosensing Methods Based on Different Isothermal Amplification Strategies: A Case Study with Erwinia amylovora
title_full_unstemmed Comparison of Biosensing Methods Based on Different Isothermal Amplification Strategies: A Case Study with Erwinia amylovora
title_short Comparison of Biosensing Methods Based on Different Isothermal Amplification Strategies: A Case Study with Erwinia amylovora
title_sort comparison of biosensing methods based on different isothermal amplification strategies: a case study with erwinia amylovora
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9776058/
https://www.ncbi.nlm.nih.gov/pubmed/36551141
http://dx.doi.org/10.3390/bios12121174
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