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A rapid DNA extraction and loop-mediated isothermal amplification (LAMP) procedure was developed and evaluated for the detection of two specific groups of phytoplasmas from infected plant material. Primers based upon the 16-23S intergenic spacer (IGS) region were evaluated in LAMP assays for amplification of group 16SrI (aster yellows group) and group 16SrXXII (Cape St Paul wilt group) phytoplasma strains. DNA could be extracted from leaf material (16SrI phytoplasmas) or coconut trunk borings (16SrXXII phytoplasmas) onto the membranes of lateral flow devices, and small sections of these membranes were then added directly into the LAMP reaction mixture and incubated for 45 min at 65°C. Positive reactions were detected through the hydroxyl napthol blue colorimetric assay within 1 h of the start of DNA extraction, and were confirmed by subsequent agarose gel electrophoresis of the LAMP products. The level of detection was comparable to that obtained by nested PCR using conventional 16S rDNA phytoplasma-specific primers. Furthermore, the assays were specific for the phytoplasmas they were designed to detect - the 16SrI assay only detected 16SrI phytoplasmas and not those from any other phylogenetic groups, whilst the 16SrXXII assay only detected 16SrXXII phytoplasmas. The DNA extractions and LAMP assay are easy to perform, requiring minimal equipment, and may therefore form the basis of a rapid and reliable field-detection system for phytoplasmas. © 2010 The Authors.
Author(s): Tomlinson JA, Boonham N, Dickinson M
Publication type: Article
Publication status: Published
Journal: Plant Pathology
Year: 2010
Volume: 59
Issue: 3
Pages: 465-471
Print publication date: 01/06/2010
Online publication date: 12/01/2010
ISSN (print): 0032-0862
ISSN (electronic): 1365-3059
Publisher: Wiley-Blackwell
URL: https://doi.org/10.1111/j.1365-3059.2009.02233.x
DOI: 10.1111/j.1365-3059.2009.02233.x
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