کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
5534466 1551168 2017 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
General phytoplasma detection by a q-PCR method using mycoplasma primers
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی بیولوژی سلول
پیش نمایش صفحه اول مقاله
General phytoplasma detection by a q-PCR method using mycoplasma primers
چکیده انگلیسی


- 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.

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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Molecular and Cellular Probes - Volume 35, October 2017, Pages 1-7
نویسندگان
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