General phytoplasma detection by a q-PCR method using mycoplasma primers. (October 2017) - Satta, Eleonora
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 10 6 to 10 3 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. Highlights: A general qPCR to detect several phytoplasma ribosomal groups has been developed. A primer pair designed for mycoplasmas also amplify phytoplasmas. The melting temperature allows to discriminate between mollicute and non-mollicute.