Test Performance Study on qPCR Assays for Detection of <i>Phyllosticta citricarpa</i>
Citrus black spot (CBS), caused by the fungus <i>Phyllosticta citricarpa</i>, significantly affects citrus fruit marketability and can lead to premature fruit drop. Accurate and reliable detection of this quarantine pathogen is crucial, particularly for asymptomatic plant material. This...
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MDPI AG
2025-04-01
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| author | Tjaša Jakomin Janja Zajc Žunič Polona Kogovšek |
| author_facet | Tjaša Jakomin Janja Zajc Žunič Polona Kogovšek |
| author_sort | Tjaša Jakomin |
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| description | Citrus black spot (CBS), caused by the fungus <i>Phyllosticta citricarpa</i>, significantly affects citrus fruit marketability and can lead to premature fruit drop. Accurate and reliable detection of this quarantine pathogen is crucial, particularly for asymptomatic plant material. This study evaluated two qPCR assays, the EPPO recommended assay PC and assay Pc-TEF1, based on <i>TEF</i> region, for detecting <i>P. citricarpa</i> through a collaborative test performance study (TPS). DNA from the isolates of <i>Phyllosticta</i> spp. and other fungi was spiked into citrus fruit peel extracts (lemon, orange, and pomelo) and distributed among 13 laboratories. Sample and qPCR assay stability under typical transport conditions was confirmed, although prolonged storage affected Pc-TEF1 assay performance. The assays were assessed based on sensitivity, specificity, reproducibility, and repeatability. Both assays demonstrated high performance, with repeatability and reproducibility exceeding 95%. The PC assay, as expected, detected different related <i>Phyllosticta</i> species, while Pc-TEF1 showed higher specificity for <i>P. citricarpa</i> included in the TPS alone. Additionally, inhibitory effects were observed specifically in the pomelo peel samples, suggesting matrix-dependent variability. This TPS confirms that both PC and Pc-TEF1 qPCR assays are robust. Further evaluation of the qPCR assays would support the selection of the most reliable assays for the detection of <i>P. citricarpa</i>, contributing to the effective management of CBS disease in citrus production and trade. |
| format | Article |
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| institution | Kabale University |
| issn | 2076-0817 |
| language | English |
| publishDate | 2025-04-01 |
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| series | Pathogens |
| spelling | doaj-art-e6d56e073f784e7eb4090e56a37fbb992025-08-20T03:48:02ZengMDPI AGPathogens2076-08172025-04-0114541310.3390/pathogens14050413Test Performance Study on qPCR Assays for Detection of <i>Phyllosticta citricarpa</i>Tjaša Jakomin0Janja Zajc Žunič1Polona Kogovšek2Department of Biotechnology and Systems Biology, National Institute of Biology, 1000 Ljubljana, SloveniaPlant Protection Department, Agricultural Institute of Slovenia, 1000 Ljubljana, SloveniaDepartment of Biotechnology and Systems Biology, National Institute of Biology, 1000 Ljubljana, SloveniaCitrus black spot (CBS), caused by the fungus <i>Phyllosticta citricarpa</i>, significantly affects citrus fruit marketability and can lead to premature fruit drop. Accurate and reliable detection of this quarantine pathogen is crucial, particularly for asymptomatic plant material. This study evaluated two qPCR assays, the EPPO recommended assay PC and assay Pc-TEF1, based on <i>TEF</i> region, for detecting <i>P. citricarpa</i> through a collaborative test performance study (TPS). DNA from the isolates of <i>Phyllosticta</i> spp. and other fungi was spiked into citrus fruit peel extracts (lemon, orange, and pomelo) and distributed among 13 laboratories. Sample and qPCR assay stability under typical transport conditions was confirmed, although prolonged storage affected Pc-TEF1 assay performance. The assays were assessed based on sensitivity, specificity, reproducibility, and repeatability. Both assays demonstrated high performance, with repeatability and reproducibility exceeding 95%. The PC assay, as expected, detected different related <i>Phyllosticta</i> species, while Pc-TEF1 showed higher specificity for <i>P. citricarpa</i> included in the TPS alone. Additionally, inhibitory effects were observed specifically in the pomelo peel samples, suggesting matrix-dependent variability. This TPS confirms that both PC and Pc-TEF1 qPCR assays are robust. Further evaluation of the qPCR assays would support the selection of the most reliable assays for the detection of <i>P. citricarpa</i>, contributing to the effective management of CBS disease in citrus production and trade.https://www.mdpi.com/2076-0817/14/5/413test performance study<i>Phyllosticta citricarpa</i>real time PCR<i>TEF1</i> |
| spellingShingle | Tjaša Jakomin Janja Zajc Žunič Polona Kogovšek Test Performance Study on qPCR Assays for Detection of <i>Phyllosticta citricarpa</i> Pathogens test performance study <i>Phyllosticta citricarpa</i> real time PCR <i>TEF1</i> |
| title | Test Performance Study on qPCR Assays for Detection of <i>Phyllosticta citricarpa</i> |
| title_full | Test Performance Study on qPCR Assays for Detection of <i>Phyllosticta citricarpa</i> |
| title_fullStr | Test Performance Study on qPCR Assays for Detection of <i>Phyllosticta citricarpa</i> |
| title_full_unstemmed | Test Performance Study on qPCR Assays for Detection of <i>Phyllosticta citricarpa</i> |
| title_short | Test Performance Study on qPCR Assays for Detection of <i>Phyllosticta citricarpa</i> |
| title_sort | test performance study on qpcr assays for detection of i phyllosticta citricarpa i |
| topic | test performance study <i>Phyllosticta citricarpa</i> real time PCR <i>TEF1</i> |
| url | https://www.mdpi.com/2076-0817/14/5/413 |
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