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Interlaboratory Performance of a Real-Time PCR Method for Detection of Ceratocystis platani, the Agent of Canker Stain of Platanus spp.
Ceratocystis platani (CP), an ascomycetous fungus, is the agent of canker stain, a lethal vascular disease of Platanus species. Ceratocystis platani has been listed as a quarantine pest (EPPO A2 list) due to extensive damage caused in Southern Europe and the Mediterranean region. As traditional diag...
Autores principales: | , , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9330143/ https://www.ncbi.nlm.nih.gov/pubmed/35893146 http://dx.doi.org/10.3390/jof8080778 |
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author | Brunetti, Angela Heungens, Kurt Hubert, Jacqueline Ioos, Renaud Bianchi, Gian Luca De Amicis, Francesca Chandelier, Anne Van Der Linde, Sietse Perez-Sierra, Ana Gualandri, Valeria Silletti, Maria Rosaria Trisciuzzi, Vito Nicola Rimondi, Silvia Baschieri, Tiziana Romano, Elio Lumia, Valentina Luigi, Marta Faggioli, Francesco Pilotti, Massimo |
author_facet | Brunetti, Angela Heungens, Kurt Hubert, Jacqueline Ioos, Renaud Bianchi, Gian Luca De Amicis, Francesca Chandelier, Anne Van Der Linde, Sietse Perez-Sierra, Ana Gualandri, Valeria Silletti, Maria Rosaria Trisciuzzi, Vito Nicola Rimondi, Silvia Baschieri, Tiziana Romano, Elio Lumia, Valentina Luigi, Marta Faggioli, Francesco Pilotti, Massimo |
author_sort | Brunetti, Angela |
collection | PubMed |
description | Ceratocystis platani (CP), an ascomycetous fungus, is the agent of canker stain, a lethal vascular disease of Platanus species. Ceratocystis platani has been listed as a quarantine pest (EPPO A2 list) due to extensive damage caused in Southern Europe and the Mediterranean region. As traditional diagnostic assays are ineffective, a Real-Time PCR detection method based on EvaGreen, SYBR Green, and Taqman assays was previously developed, validated in-house, and included in the official EPPO standard PM7/14 (2). Here, we describe the results of a test performance study performed by nine European laboratories for the purpose of an interlaboratory validation. Verification of the DNA extracted from biological samples guaranteed the high quality of preparations, and the stability and the homogeneity of the aliquots intended for the laboratories. All of the laboratories reproduced nearly identical standard curves with efficiencies close to 100%. Testing of blind-coded DNA extracted from wood samples revealed that all performance parameters—diagnostic sensitivity, diagnostic specificity, accuracy and reproducibility—were best fit in most cases both at the laboratory and at the assay level. The previously established limit of detection, 3 fg per PCR reaction, was also validated with similar excellent results. The high interlaboratory performance of this Real-Time PCR method confirms its value as a primary tool to safeguard C. platani-free countries by way of an accurate monitoring, and to investigate the resistance level of potentially canker stain-resistant Platanus genotypes. |
format | Online Article Text |
id | pubmed-9330143 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-93301432022-07-29 Interlaboratory Performance of a Real-Time PCR Method for Detection of Ceratocystis platani, the Agent of Canker Stain of Platanus spp. Brunetti, Angela Heungens, Kurt Hubert, Jacqueline Ioos, Renaud Bianchi, Gian Luca De Amicis, Francesca Chandelier, Anne Van Der Linde, Sietse Perez-Sierra, Ana Gualandri, Valeria Silletti, Maria Rosaria Trisciuzzi, Vito Nicola Rimondi, Silvia Baschieri, Tiziana Romano, Elio Lumia, Valentina Luigi, Marta Faggioli, Francesco Pilotti, Massimo J Fungi (Basel) Article Ceratocystis platani (CP), an ascomycetous fungus, is the agent of canker stain, a lethal vascular disease of Platanus species. Ceratocystis platani has been listed as a quarantine pest (EPPO A2 list) due to extensive damage caused in Southern Europe and the Mediterranean region. As traditional diagnostic assays are ineffective, a Real-Time PCR detection method based on EvaGreen, SYBR Green, and Taqman assays was previously developed, validated in-house, and included in the official EPPO standard PM7/14 (2). Here, we describe the results of a test performance study performed by nine European laboratories for the purpose of an interlaboratory validation. Verification of the DNA extracted from biological samples guaranteed the high quality of preparations, and the stability and the homogeneity of the aliquots intended for the laboratories. All of the laboratories reproduced nearly identical standard curves with efficiencies close to 100%. Testing of blind-coded DNA extracted from wood samples revealed that all performance parameters—diagnostic sensitivity, diagnostic specificity, accuracy and reproducibility—were best fit in most cases both at the laboratory and at the assay level. The previously established limit of detection, 3 fg per PCR reaction, was also validated with similar excellent results. The high interlaboratory performance of this Real-Time PCR method confirms its value as a primary tool to safeguard C. platani-free countries by way of an accurate monitoring, and to investigate the resistance level of potentially canker stain-resistant Platanus genotypes. MDPI 2022-07-26 /pmc/articles/PMC9330143/ /pubmed/35893146 http://dx.doi.org/10.3390/jof8080778 Text en © 2022 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 Brunetti, Angela Heungens, Kurt Hubert, Jacqueline Ioos, Renaud Bianchi, Gian Luca De Amicis, Francesca Chandelier, Anne Van Der Linde, Sietse Perez-Sierra, Ana Gualandri, Valeria Silletti, Maria Rosaria Trisciuzzi, Vito Nicola Rimondi, Silvia Baschieri, Tiziana Romano, Elio Lumia, Valentina Luigi, Marta Faggioli, Francesco Pilotti, Massimo Interlaboratory Performance of a Real-Time PCR Method for Detection of Ceratocystis platani, the Agent of Canker Stain of Platanus spp. |
title | Interlaboratory Performance of a Real-Time PCR Method for Detection of Ceratocystis platani, the Agent of Canker Stain of Platanus spp. |
title_full | Interlaboratory Performance of a Real-Time PCR Method for Detection of Ceratocystis platani, the Agent of Canker Stain of Platanus spp. |
title_fullStr | Interlaboratory Performance of a Real-Time PCR Method for Detection of Ceratocystis platani, the Agent of Canker Stain of Platanus spp. |
title_full_unstemmed | Interlaboratory Performance of a Real-Time PCR Method for Detection of Ceratocystis platani, the Agent of Canker Stain of Platanus spp. |
title_short | Interlaboratory Performance of a Real-Time PCR Method for Detection of Ceratocystis platani, the Agent of Canker Stain of Platanus spp. |
title_sort | interlaboratory performance of a real-time pcr method for detection of ceratocystis platani, the agent of canker stain of platanus spp. |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9330143/ https://www.ncbi.nlm.nih.gov/pubmed/35893146 http://dx.doi.org/10.3390/jof8080778 |
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