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The Challenge for Rapid Detection of High-Structured Circular RNA: Assay of Potato Spindle Tuber Viroid Based on Recombinase Polymerase Amplification and Lateral Flow Tests

An assay was developed to detect the potato spindle tuber viroid (PSTVd), a dangerous plant pathogen that causes crop damage resulting in economic losses in the potato agriculture sector. The assay was based on the reverse transcription and recombinase polymerase amplification (RT-RPA) of PSTVd RNA...

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Autores principales: Ivanov, Aleksandr V., Shmyglya, Irina V., Zherdev, Anatoly V., Dzantiev, Boris B., Safenkova, Irina V.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7650583/
https://www.ncbi.nlm.nih.gov/pubmed/33076508
http://dx.doi.org/10.3390/plants9101369
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author Ivanov, Aleksandr V.
Shmyglya, Irina V.
Zherdev, Anatoly V.
Dzantiev, Boris B.
Safenkova, Irina V.
author_facet Ivanov, Aleksandr V.
Shmyglya, Irina V.
Zherdev, Anatoly V.
Dzantiev, Boris B.
Safenkova, Irina V.
author_sort Ivanov, Aleksandr V.
collection PubMed
description An assay was developed to detect the potato spindle tuber viroid (PSTVd), a dangerous plant pathogen that causes crop damage resulting in economic losses in the potato agriculture sector. The assay was based on the reverse transcription and recombinase polymerase amplification (RT-RPA) of PSTVd RNA coupled with amplicon detection via lateral flow assay (LFA). Primers labeled with fluorescein and biotin were designed for RT-RPA for effective recognition of the loop regions in the high-structured circular RNA of PSTVd. The labeled DNA amplicon was detected using lateral flow test strips consisting of a conjugate of gold nanoparticles with antibodies specific to fluorescein and streptavidin in the test zone. The RT-RPA-LFA detected 10(6) copies of in vitro transcribed PSTVd RNA in reaction or up to 1:10(7) diluted extracts of infected plant leaves. The assay took 30 min, including the RT-RPA stage and the LFA stage. The testing of healthy and infected potato samples showed full concordance between the developed RT-RPA-LFA and quantitative reverse transcription polymerase chain reaction (RT-qPCR) and the commercial kit. The obtained results proved the feasibility of using the developed assay to detect PSTVd from a natural source.
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spelling pubmed-76505832020-11-10 The Challenge for Rapid Detection of High-Structured Circular RNA: Assay of Potato Spindle Tuber Viroid Based on Recombinase Polymerase Amplification and Lateral Flow Tests Ivanov, Aleksandr V. Shmyglya, Irina V. Zherdev, Anatoly V. Dzantiev, Boris B. Safenkova, Irina V. Plants (Basel) Article An assay was developed to detect the potato spindle tuber viroid (PSTVd), a dangerous plant pathogen that causes crop damage resulting in economic losses in the potato agriculture sector. The assay was based on the reverse transcription and recombinase polymerase amplification (RT-RPA) of PSTVd RNA coupled with amplicon detection via lateral flow assay (LFA). Primers labeled with fluorescein and biotin were designed for RT-RPA for effective recognition of the loop regions in the high-structured circular RNA of PSTVd. The labeled DNA amplicon was detected using lateral flow test strips consisting of a conjugate of gold nanoparticles with antibodies specific to fluorescein and streptavidin in the test zone. The RT-RPA-LFA detected 10(6) copies of in vitro transcribed PSTVd RNA in reaction or up to 1:10(7) diluted extracts of infected plant leaves. The assay took 30 min, including the RT-RPA stage and the LFA stage. The testing of healthy and infected potato samples showed full concordance between the developed RT-RPA-LFA and quantitative reverse transcription polymerase chain reaction (RT-qPCR) and the commercial kit. The obtained results proved the feasibility of using the developed assay to detect PSTVd from a natural source. MDPI 2020-10-15 /pmc/articles/PMC7650583/ /pubmed/33076508 http://dx.doi.org/10.3390/plants9101369 Text en © 2020 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 (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Ivanov, Aleksandr V.
Shmyglya, Irina V.
Zherdev, Anatoly V.
Dzantiev, Boris B.
Safenkova, Irina V.
The Challenge for Rapid Detection of High-Structured Circular RNA: Assay of Potato Spindle Tuber Viroid Based on Recombinase Polymerase Amplification and Lateral Flow Tests
title The Challenge for Rapid Detection of High-Structured Circular RNA: Assay of Potato Spindle Tuber Viroid Based on Recombinase Polymerase Amplification and Lateral Flow Tests
title_full The Challenge for Rapid Detection of High-Structured Circular RNA: Assay of Potato Spindle Tuber Viroid Based on Recombinase Polymerase Amplification and Lateral Flow Tests
title_fullStr The Challenge for Rapid Detection of High-Structured Circular RNA: Assay of Potato Spindle Tuber Viroid Based on Recombinase Polymerase Amplification and Lateral Flow Tests
title_full_unstemmed The Challenge for Rapid Detection of High-Structured Circular RNA: Assay of Potato Spindle Tuber Viroid Based on Recombinase Polymerase Amplification and Lateral Flow Tests
title_short The Challenge for Rapid Detection of High-Structured Circular RNA: Assay of Potato Spindle Tuber Viroid Based on Recombinase Polymerase Amplification and Lateral Flow Tests
title_sort challenge for rapid detection of high-structured circular rna: assay of potato spindle tuber viroid based on recombinase polymerase amplification and lateral flow tests
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7650583/
https://www.ncbi.nlm.nih.gov/pubmed/33076508
http://dx.doi.org/10.3390/plants9101369
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