|Title:||General phytoplasma detection by a q-PCR method using mycoplasma primers||Authors:||Satta, E.
Nanni, I. M.
Poveda, José B.
Ramírez, Ana S.
|UNESCO Clasification:||32 Ciencias médicas||Keywords:||Mollicutes
|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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