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Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer
Early detection of infection is very important for efficient management of Mycosphaerella graminicola leaf blotch. To monitor and quantify the occurrence of this fungus during the growing season, a diagnostic method based on real-time PCR was developed. Standard and real-time PCR assays were develop...
Autores principales: | , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
Molecular Diversity Preservation International (MDPI)
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3039974/ https://www.ncbi.nlm.nih.gov/pubmed/21340008 http://dx.doi.org/10.3390/ijms12010682 |
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author | Abd-Elsalam, Kamel Bahkali, Ali H. Moslem, Mohamed De Wit, Pierre J. G. M. Verreet, Joseph-Alexander |
author_facet | Abd-Elsalam, Kamel Bahkali, Ali H. Moslem, Mohamed De Wit, Pierre J. G. M. Verreet, Joseph-Alexander |
author_sort | Abd-Elsalam, Kamel |
collection | PubMed |
description | Early detection of infection is very important for efficient management of Mycosphaerella graminicola leaf blotch. To monitor and quantify the occurrence of this fungus during the growing season, a diagnostic method based on real-time PCR was developed. Standard and real-time PCR assays were developed using SYBR Green chemistry to quantify M. graminicola in vitro or in wheat samples. Microsatellite dinucleotide specific-primers were designed based on microsatellite repeats of sequences present in the genome of M. graminicola. Specificity was checked by analyzing DNA of 55 M. graminicola isolates obtained from different geographical origins. The method appears to be highly specific for detecting M. graminicola; no fluorescent signals were observed from 14 other closely related taxa. Primer (CT) 7 G amplified a specific amplicon of 570 bp from all M. graminicola isolates. The primers did not amplify DNA extracted from 14 other fungal species. The approximate melting temperature (Tm) of the (CT) 7 G primer was 84.2 °C. The detection limit of the real-time PCR assay with the primer sets (CT) 7 G is 10 fg/25 μL, as compared to 10 pg/25 μL using conventional PCR technology. From symptomless leaves, a PCR fragment could be generated two days after inoculation. Both conventional and real-time PCR could successfully detect the fungus from artificially inoculated wheat leaves. However, real-time PCR appeared much more sensitive than conventional PCR. The developed quantitative real-time PCR method proved to be rapid, sensitive, specific, cost-effective and reliable for the identification and quantification of M. graminicola in wheat. |
format | Text |
id | pubmed-3039974 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | Molecular Diversity Preservation International (MDPI) |
record_format | MEDLINE/PubMed |
spelling | pubmed-30399742011-02-18 Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer Abd-Elsalam, Kamel Bahkali, Ali H. Moslem, Mohamed De Wit, Pierre J. G. M. Verreet, Joseph-Alexander Int J Mol Sci Article Early detection of infection is very important for efficient management of Mycosphaerella graminicola leaf blotch. To monitor and quantify the occurrence of this fungus during the growing season, a diagnostic method based on real-time PCR was developed. Standard and real-time PCR assays were developed using SYBR Green chemistry to quantify M. graminicola in vitro or in wheat samples. Microsatellite dinucleotide specific-primers were designed based on microsatellite repeats of sequences present in the genome of M. graminicola. Specificity was checked by analyzing DNA of 55 M. graminicola isolates obtained from different geographical origins. The method appears to be highly specific for detecting M. graminicola; no fluorescent signals were observed from 14 other closely related taxa. Primer (CT) 7 G amplified a specific amplicon of 570 bp from all M. graminicola isolates. The primers did not amplify DNA extracted from 14 other fungal species. The approximate melting temperature (Tm) of the (CT) 7 G primer was 84.2 °C. The detection limit of the real-time PCR assay with the primer sets (CT) 7 G is 10 fg/25 μL, as compared to 10 pg/25 μL using conventional PCR technology. From symptomless leaves, a PCR fragment could be generated two days after inoculation. Both conventional and real-time PCR could successfully detect the fungus from artificially inoculated wheat leaves. However, real-time PCR appeared much more sensitive than conventional PCR. The developed quantitative real-time PCR method proved to be rapid, sensitive, specific, cost-effective and reliable for the identification and quantification of M. graminicola in wheat. Molecular Diversity Preservation International (MDPI) 2011-01-19 /pmc/articles/PMC3039974/ /pubmed/21340008 http://dx.doi.org/10.3390/ijms12010682 Text en © 2011 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. http://creativecommons.org/licenses/by/3.0 This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). |
spellingShingle | Article Abd-Elsalam, Kamel Bahkali, Ali H. Moslem, Mohamed De Wit, Pierre J. G. M. Verreet, Joseph-Alexander Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer |
title | Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer |
title_full | Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer |
title_fullStr | Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer |
title_full_unstemmed | Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer |
title_short | Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer |
title_sort | detection of mycosphaerella graminicola in wheat leaves by a microsatellite dinucleotide specific-primer |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3039974/ https://www.ncbi.nlm.nih.gov/pubmed/21340008 http://dx.doi.org/10.3390/ijms12010682 |
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