One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees
A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total n...
| Autores principales: | , , , |
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| Formato: | Artículo |
| Lenguaje: | Inglés |
| Publicado: |
2017
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| Acceso en línea: | http://hdl.handle.net/20.500.11939/5556 |
| _version_ | 1855491944516419584 |
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| author | Malandraki, Ioanna Varveri, Christina Olmos, Antonio Vassilakos, Nikon |
| author_browse | Malandraki, Ioanna Olmos, Antonio Varveri, Christina Vassilakos, Nikon |
| author_facet | Malandraki, Ioanna Varveri, Christina Olmos, Antonio Vassilakos, Nikon |
| author_sort | Malandraki, Ioanna |
| collection | ReDivia |
| description | A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10(-5) for viroids and 10(-4) for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys. (C) 2014 Elsevier B.V. All rights reserved. |
| format | Artículo |
| id | ReDivia5556 |
| institution | Instituto Valenciano de Investigaciones Agrarias (IVIA) |
| language | Inglés |
| publishDate | 2017 |
| publishDateRange | 2017 |
| publishDateSort | 2017 |
| record_format | dspace |
| spelling | ReDivia55562025-04-25T14:43:17Z One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees Malandraki, Ioanna Varveri, Christina Olmos, Antonio Vassilakos, Nikon Real-time PCR MGB probes Apple scar skin viroid Pear blister canker viroid 'Candidatus Phytoplasma mali' 'Candidatus Phytoplasma pyri' POLYMERASE-CHAIN-REACTION REACTION ASSAYS IDENTIFICATION SEQUENCE VIRUSES DIFFERENTIATION VALIDATION ORGANISMS PLANTS A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10(-5) for viroids and 10(-4) for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys. (C) 2014 Elsevier B.V. All rights reserved. 2017-06-01T10:12:33Z 2017-06-01T10:12:33Z 2015 MAR 1 article Malandraki, Ioanna, Varveri, Christina, Olmos, A., Vassilakos, Nikon (2015). One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees. Journal of virological methods, 213, 12-17. 0166-0934 http://hdl.handle.net/20.500.11939/5556 10.1016/j.jviromet.2014.11.010 en openAccess Impreso |
| spellingShingle | Real-time PCR MGB probes Apple scar skin viroid Pear blister canker viroid 'Candidatus Phytoplasma mali' 'Candidatus Phytoplasma pyri' POLYMERASE-CHAIN-REACTION REACTION ASSAYS IDENTIFICATION SEQUENCE VIRUSES DIFFERENTIATION VALIDATION ORGANISMS PLANTS Malandraki, Ioanna Varveri, Christina Olmos, Antonio Vassilakos, Nikon One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees |
| title | One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees |
| title_full | One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees |
| title_fullStr | One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees |
| title_full_unstemmed | One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees |
| title_short | One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees |
| title_sort | one step multiplex quantitative rt pcr for the simultaneous detection of viroids and phytoplasmas of pome fruit trees |
| topic | Real-time PCR MGB probes Apple scar skin viroid Pear blister canker viroid 'Candidatus Phytoplasma mali' 'Candidatus Phytoplasma pyri' POLYMERASE-CHAIN-REACTION REACTION ASSAYS IDENTIFICATION SEQUENCE VIRUSES DIFFERENTIATION VALIDATION ORGANISMS PLANTS |
| url | http://hdl.handle.net/20.500.11939/5556 |
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