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Title: | General phytoplasma detection by a q-PCR method using mycoplasma primers | Authors: | Satta, E. Nanni, I. M. Contaldo, N. Collina, M. Poveda, José B. Ramírez, Ana S. Bertaccini, A. |
UNESCO Clasification: | 32 Ciencias médicas | Keywords: | Mollicutes Prokaryotes Diagnostic Plant diseases Quantification |
Issue Date: | 2017 | Journal: | Molecular and Cellular Probes | Abstract: | Phytoplasmas and mycoplasmas are bacteria belonging to the class Mollicutes. In this study, a fine tuning of quantitative polymerase chain reaction (qPCR) with a universal mycoplasma primer pair (GPO3F/MGSO) targeting the 16S rRNA gene was carried out on phytoplasmas. The dissociation curves of DNAs from Catharanthus roseus phytoplasma-infected micropropagated shoots and from phytoplasma field-infected plant samples showed a single peak at 82.5 °C (±0.5) specifically detecting phytoplasmas belonging to several ribosomal groups. Assay specificity was determined with DNA of selected bacteria: ‘Candidatus Liberibacter solanacearum’, Xylella fastidiosa, Ralstonia solanacearum and Clavibacter michiganensis. No amplification curves were observed with any of these tested bacteria except ‘Ca. L. solanacearum’ that was amplified with a melting temperature at 85 °C. Absolute quantification of phytoplasma titer was calculated using standard curves prepared from serial dilutions of plasmids containing the cloned fragment GPO3F/MGSO from European stone fruit yellows phytoplasma. Phytoplasma copy number ranged from 106 to 103 according with the sample. The sensitivity evaluated comparing plasmid serial dilutions resulted 10−6 for conventional PCR and 10−7 for qPCR. The latter method resulted therefore able to detect very low concentrations of phytoplasma in plant material. | URI: | http://hdl.handle.net/10553/37180 | ISSN: | 0890-8508 | DOI: | 10.1016/j.mcp.2017.05.008 | Source: | Molecular and Cellular Probes[ISSN 0890-8508],v. 35, p. 1-7 |
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