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Response Surface Methodology for Optimization of Multiplex-PCR Protocols for Detection of TYLCV, TSWV and Fol Molecular Markers: Analytical Performance Evaluation

Molecular markers linked to disease resistance genes which affect economically important crops are of great interest. In the case of tomato, a major focus on resistance breeding to multiple fungal and viral pathogens such as Tomato yellow leaf curl virus (TYLCV), Tomato spotted wilt virus (TSWV) and...

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Autores principales: Lafrance, Richecarde, Valdez-Torres, José Benigno, Villicaña, Claudia, García-Estrada, Raymundo Saúl, Esparza-Araiza, Mayra Janeth, León-Félix, Josefina
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9956084/
https://www.ncbi.nlm.nih.gov/pubmed/36833262
http://dx.doi.org/10.3390/genes14020337
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author Lafrance, Richecarde
Valdez-Torres, José Benigno
Villicaña, Claudia
García-Estrada, Raymundo Saúl
Esparza-Araiza, Mayra Janeth
León-Félix, Josefina
author_facet Lafrance, Richecarde
Valdez-Torres, José Benigno
Villicaña, Claudia
García-Estrada, Raymundo Saúl
Esparza-Araiza, Mayra Janeth
León-Félix, Josefina
author_sort Lafrance, Richecarde
collection PubMed
description Molecular markers linked to disease resistance genes which affect economically important crops are of great interest. In the case of tomato, a major focus on resistance breeding to multiple fungal and viral pathogens such as Tomato yellow leaf curl virus (TYLCV), Tomato spotted wilt virus (TSWV) and Fusarium oxysporum f. sp. lycopersici (Fol), have led to the introgression of several resistance genes; therefore, molecular markers have become important in molecular-assisted selection (MAS) of tomato varieties resistant to those pathogens. However, assays that allow simultaneous evaluation of resistant genotypes, such as multiplex PCR, need to be optimized and evaluated to demonstrate their analytical performance, as many factors can affect them. This work aimed to generate multiplex PCR protocols for the joint detection of the molecular markers associated with pathogen resistance genes in tomato plants that are sensitive, specific and repeatable. For the optimization a central composite design of a response surface methodology (RSM-CCD) was used. For analytical performance evaluation, specificity/selectivity and sensibility (limit of detection and dynamic range) were analyzed. Two protocols were optimized: the first one with a desirability of 1.00, contained two markers (At-2 and P7-43) linked to I- and I-3-resistant genes. The second one with a desirability of 0.99, contained markers (SSR-67, SW5 and P6-25) linked to I-, Sw-5-, and Ty-3-resistant genes. For protocol 1, all the commercial hybrids (7/7) were resistant to Fol, and for protocol 2, two hybrids were resistant to Fol, one to TSWV and one to TYLCV with good analytical performance. In both protocols, the varieties considered susceptible to the pathogens, no-amplicon or susceptible amplicons, were observed. The optimized multiplex PCR protocols showed dynamic ranges from 5.97 up to 161.3 ng DNA. The limit of detection was 17.92 ng and 53.76 ng DNA for protocols 1 and 2, respectively, giving 100% positive results in the test replicates. This method allowed to develop optimized multiplex PCR protocols with few assays which translates into less time and resources, without sacrificing method performance.
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spelling pubmed-99560842023-02-25 Response Surface Methodology for Optimization of Multiplex-PCR Protocols for Detection of TYLCV, TSWV and Fol Molecular Markers: Analytical Performance Evaluation Lafrance, Richecarde Valdez-Torres, José Benigno Villicaña, Claudia García-Estrada, Raymundo Saúl Esparza-Araiza, Mayra Janeth León-Félix, Josefina Genes (Basel) Article Molecular markers linked to disease resistance genes which affect economically important crops are of great interest. In the case of tomato, a major focus on resistance breeding to multiple fungal and viral pathogens such as Tomato yellow leaf curl virus (TYLCV), Tomato spotted wilt virus (TSWV) and Fusarium oxysporum f. sp. lycopersici (Fol), have led to the introgression of several resistance genes; therefore, molecular markers have become important in molecular-assisted selection (MAS) of tomato varieties resistant to those pathogens. However, assays that allow simultaneous evaluation of resistant genotypes, such as multiplex PCR, need to be optimized and evaluated to demonstrate their analytical performance, as many factors can affect them. This work aimed to generate multiplex PCR protocols for the joint detection of the molecular markers associated with pathogen resistance genes in tomato plants that are sensitive, specific and repeatable. For the optimization a central composite design of a response surface methodology (RSM-CCD) was used. For analytical performance evaluation, specificity/selectivity and sensibility (limit of detection and dynamic range) were analyzed. Two protocols were optimized: the first one with a desirability of 1.00, contained two markers (At-2 and P7-43) linked to I- and I-3-resistant genes. The second one with a desirability of 0.99, contained markers (SSR-67, SW5 and P6-25) linked to I-, Sw-5-, and Ty-3-resistant genes. For protocol 1, all the commercial hybrids (7/7) were resistant to Fol, and for protocol 2, two hybrids were resistant to Fol, one to TSWV and one to TYLCV with good analytical performance. In both protocols, the varieties considered susceptible to the pathogens, no-amplicon or susceptible amplicons, were observed. The optimized multiplex PCR protocols showed dynamic ranges from 5.97 up to 161.3 ng DNA. The limit of detection was 17.92 ng and 53.76 ng DNA for protocols 1 and 2, respectively, giving 100% positive results in the test replicates. This method allowed to develop optimized multiplex PCR protocols with few assays which translates into less time and resources, without sacrificing method performance. MDPI 2023-01-28 /pmc/articles/PMC9956084/ /pubmed/36833262 http://dx.doi.org/10.3390/genes14020337 Text en © 2023 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
Lafrance, Richecarde
Valdez-Torres, José Benigno
Villicaña, Claudia
García-Estrada, Raymundo Saúl
Esparza-Araiza, Mayra Janeth
León-Félix, Josefina
Response Surface Methodology for Optimization of Multiplex-PCR Protocols for Detection of TYLCV, TSWV and Fol Molecular Markers: Analytical Performance Evaluation
title Response Surface Methodology for Optimization of Multiplex-PCR Protocols for Detection of TYLCV, TSWV and Fol Molecular Markers: Analytical Performance Evaluation
title_full Response Surface Methodology for Optimization of Multiplex-PCR Protocols for Detection of TYLCV, TSWV and Fol Molecular Markers: Analytical Performance Evaluation
title_fullStr Response Surface Methodology for Optimization of Multiplex-PCR Protocols for Detection of TYLCV, TSWV and Fol Molecular Markers: Analytical Performance Evaluation
title_full_unstemmed Response Surface Methodology for Optimization of Multiplex-PCR Protocols for Detection of TYLCV, TSWV and Fol Molecular Markers: Analytical Performance Evaluation
title_short Response Surface Methodology for Optimization of Multiplex-PCR Protocols for Detection of TYLCV, TSWV and Fol Molecular Markers: Analytical Performance Evaluation
title_sort response surface methodology for optimization of multiplex-pcr protocols for detection of tylcv, tswv and fol molecular markers: analytical performance evaluation
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9956084/
https://www.ncbi.nlm.nih.gov/pubmed/36833262
http://dx.doi.org/10.3390/genes14020337
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