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In-silico prediction of RT-qPCR-high resolution melting for broad detection of emaraviruses

Twenty-four species of RNA viruses contain members infecting economically important crops that are classified within the genus Emaravirus, family Fimoviridae. There are at least two other non-classified species that may be added. Some of these viruses are spreading rapidly and cause economically imp...

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Autores principales: Olmedo-Velarde, Alejandro, Ochoa-Corona, Francisco M., Larrea-Sarmiento, Adriana E., Elbeaino, Toufic, Flores, Francisco
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10166557/
https://www.ncbi.nlm.nih.gov/pubmed/37155676
http://dx.doi.org/10.1371/journal.pone.0272980
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author Olmedo-Velarde, Alejandro
Ochoa-Corona, Francisco M.
Larrea-Sarmiento, Adriana E.
Elbeaino, Toufic
Flores, Francisco
author_facet Olmedo-Velarde, Alejandro
Ochoa-Corona, Francisco M.
Larrea-Sarmiento, Adriana E.
Elbeaino, Toufic
Flores, Francisco
author_sort Olmedo-Velarde, Alejandro
collection PubMed
description Twenty-four species of RNA viruses contain members infecting economically important crops that are classified within the genus Emaravirus, family Fimoviridae. There are at least two other non-classified species that may be added. Some of these viruses are spreading rapidly and cause economically important diseases on several crops, raising a need for a sensitive diagnostic technique for taxonomic and quarantine purposes. High-resolution melting (HRM) has shown to be reliable for the detection, discrimination, and diagnosis of several diseases of plants, animals, and humans. This research aimed to explore the ability to predict HRM outputs coupled to reverse transcription-quantitative polymerase chain reaction (RT-qPCR). To approach this goal a pair of degenerate genus-specific primers were designed for endpoint RT-PCR and RT-qPCR-HRM and the species in the genus Emaravirus were selected to framework the development of the assays. Both nucleic acid amplification methods were able to detect in-vitro several members of seven Emaravirus species with sensitivity up to one fg of cDNA. Specific parameters for in-silico prediction of the melting temperatures of each expected emaravirus amplicon are compared to the data obtained in-vitro. A very distinct isolate of the High Plains wheat mosaic virus was also detected. The high-resolution DNA melting curves of the RT-PCR products predicted in-silico using uMelt(SM) allowed saving time while designing and developing the RT-qPCR-HRM assay since the approach avoided extensive searching for optimal HRM assay regions and rounds of HRM tests in-vitro for optimization. The resultant assay provides sensitive detection and reliable diagnosis for potentially any emaravirus, including new species or strains.
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spelling pubmed-101665572023-05-09 In-silico prediction of RT-qPCR-high resolution melting for broad detection of emaraviruses Olmedo-Velarde, Alejandro Ochoa-Corona, Francisco M. Larrea-Sarmiento, Adriana E. Elbeaino, Toufic Flores, Francisco PLoS One Research Article Twenty-four species of RNA viruses contain members infecting economically important crops that are classified within the genus Emaravirus, family Fimoviridae. There are at least two other non-classified species that may be added. Some of these viruses are spreading rapidly and cause economically important diseases on several crops, raising a need for a sensitive diagnostic technique for taxonomic and quarantine purposes. High-resolution melting (HRM) has shown to be reliable for the detection, discrimination, and diagnosis of several diseases of plants, animals, and humans. This research aimed to explore the ability to predict HRM outputs coupled to reverse transcription-quantitative polymerase chain reaction (RT-qPCR). To approach this goal a pair of degenerate genus-specific primers were designed for endpoint RT-PCR and RT-qPCR-HRM and the species in the genus Emaravirus were selected to framework the development of the assays. Both nucleic acid amplification methods were able to detect in-vitro several members of seven Emaravirus species with sensitivity up to one fg of cDNA. Specific parameters for in-silico prediction of the melting temperatures of each expected emaravirus amplicon are compared to the data obtained in-vitro. A very distinct isolate of the High Plains wheat mosaic virus was also detected. The high-resolution DNA melting curves of the RT-PCR products predicted in-silico using uMelt(SM) allowed saving time while designing and developing the RT-qPCR-HRM assay since the approach avoided extensive searching for optimal HRM assay regions and rounds of HRM tests in-vitro for optimization. The resultant assay provides sensitive detection and reliable diagnosis for potentially any emaravirus, including new species or strains. Public Library of Science 2023-05-08 /pmc/articles/PMC10166557/ /pubmed/37155676 http://dx.doi.org/10.1371/journal.pone.0272980 Text en © 2023 Olmedo-Velarde 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
Olmedo-Velarde, Alejandro
Ochoa-Corona, Francisco M.
Larrea-Sarmiento, Adriana E.
Elbeaino, Toufic
Flores, Francisco
In-silico prediction of RT-qPCR-high resolution melting for broad detection of emaraviruses
title In-silico prediction of RT-qPCR-high resolution melting for broad detection of emaraviruses
title_full In-silico prediction of RT-qPCR-high resolution melting for broad detection of emaraviruses
title_fullStr In-silico prediction of RT-qPCR-high resolution melting for broad detection of emaraviruses
title_full_unstemmed In-silico prediction of RT-qPCR-high resolution melting for broad detection of emaraviruses
title_short In-silico prediction of RT-qPCR-high resolution melting for broad detection of emaraviruses
title_sort in-silico prediction of rt-qpcr-high resolution melting for broad detection of emaraviruses
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10166557/
https://www.ncbi.nlm.nih.gov/pubmed/37155676
http://dx.doi.org/10.1371/journal.pone.0272980
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