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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
Plant diseases
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.
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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