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Comprehensive Real-Time RT-PCR Assays for the Detection of Fifteen Viruses Infecting Prunus spp.

Viruses can cause economic losses in fruit trees, including Prunus spp., by reducing yield and marketable fruit. Given the genetic diversity of viruses, reliable diagnostic methods relying on PCR are critical in determining viral infection in fruit trees. This study evaluated the broad-range detecti...

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Autores principales: Diaz-Lara, Alfredo, Stevens, Kristian, Klaassen, Vicki, Golino, Deborah, Al Rwahnih, Maher
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076543/
https://www.ncbi.nlm.nih.gov/pubmed/32092932
http://dx.doi.org/10.3390/plants9020273
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author Diaz-Lara, Alfredo
Stevens, Kristian
Klaassen, Vicki
Golino, Deborah
Al Rwahnih, Maher
author_facet Diaz-Lara, Alfredo
Stevens, Kristian
Klaassen, Vicki
Golino, Deborah
Al Rwahnih, Maher
author_sort Diaz-Lara, Alfredo
collection PubMed
description Viruses can cause economic losses in fruit trees, including Prunus spp., by reducing yield and marketable fruit. Given the genetic diversity of viruses, reliable diagnostic methods relying on PCR are critical in determining viral infection in fruit trees. This study evaluated the broad-range detection capacity of currently available real-time RT-PCR assays for Prunus-infecting viruses and developed new assays when current tests were inadequate or absent. Available assays for 15 different viruses were exhaustively evaluated in silico to determine their capacity to detect virus isolates deposited in GenBank. During this evaluation, several isolates deposited since the assay was designed exhibited nucleotide mismatches in relation to the existing assay’s primer sequences. In cases where updating an existing assay was impractical, we performed a redesign with the dual goals of assay compactness and comprehensive inclusion of genetic diversity. The efficiency of each developed assay was determined by a standard curve. To validate the assay designs, we tested them against a comprehensive set of 87 positive and negative Prunus samples independently analyzed by high throughput sequencing. As a result, all the real-time RT-PCR assays described herein successfully detected the different viruses and their corresponding isolates. To further validate the new and updated assays a Prunus germplasm collection was surveyed. The sensitive and reliable detection methods described here will be used for the large-scale pathogen testing required to maintain the highest quality nursery stock.
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spelling pubmed-70765432020-03-20 Comprehensive Real-Time RT-PCR Assays for the Detection of Fifteen Viruses Infecting Prunus spp. Diaz-Lara, Alfredo Stevens, Kristian Klaassen, Vicki Golino, Deborah Al Rwahnih, Maher Plants (Basel) Article Viruses can cause economic losses in fruit trees, including Prunus spp., by reducing yield and marketable fruit. Given the genetic diversity of viruses, reliable diagnostic methods relying on PCR are critical in determining viral infection in fruit trees. This study evaluated the broad-range detection capacity of currently available real-time RT-PCR assays for Prunus-infecting viruses and developed new assays when current tests were inadequate or absent. Available assays for 15 different viruses were exhaustively evaluated in silico to determine their capacity to detect virus isolates deposited in GenBank. During this evaluation, several isolates deposited since the assay was designed exhibited nucleotide mismatches in relation to the existing assay’s primer sequences. In cases where updating an existing assay was impractical, we performed a redesign with the dual goals of assay compactness and comprehensive inclusion of genetic diversity. The efficiency of each developed assay was determined by a standard curve. To validate the assay designs, we tested them against a comprehensive set of 87 positive and negative Prunus samples independently analyzed by high throughput sequencing. As a result, all the real-time RT-PCR assays described herein successfully detected the different viruses and their corresponding isolates. To further validate the new and updated assays a Prunus germplasm collection was surveyed. The sensitive and reliable detection methods described here will be used for the large-scale pathogen testing required to maintain the highest quality nursery stock. MDPI 2020-02-19 /pmc/articles/PMC7076543/ /pubmed/32092932 http://dx.doi.org/10.3390/plants9020273 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
Diaz-Lara, Alfredo
Stevens, Kristian
Klaassen, Vicki
Golino, Deborah
Al Rwahnih, Maher
Comprehensive Real-Time RT-PCR Assays for the Detection of Fifteen Viruses Infecting Prunus spp.
title Comprehensive Real-Time RT-PCR Assays for the Detection of Fifteen Viruses Infecting Prunus spp.
title_full Comprehensive Real-Time RT-PCR Assays for the Detection of Fifteen Viruses Infecting Prunus spp.
title_fullStr Comprehensive Real-Time RT-PCR Assays for the Detection of Fifteen Viruses Infecting Prunus spp.
title_full_unstemmed Comprehensive Real-Time RT-PCR Assays for the Detection of Fifteen Viruses Infecting Prunus spp.
title_short Comprehensive Real-Time RT-PCR Assays for the Detection of Fifteen Viruses Infecting Prunus spp.
title_sort comprehensive real-time rt-pcr assays for the detection of fifteen viruses infecting prunus spp.
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076543/
https://www.ncbi.nlm.nih.gov/pubmed/32092932
http://dx.doi.org/10.3390/plants9020273
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