Automated DNA Extraction for Large Numbers of Plant Samples

Abstract

The method described here is a rapid, total DNA extraction procedure applicable to a large number of plant samples requiring pathogen detection. The procedure combines a simple and quick homogenization step of crude extracts with DNA extraction based upon the binding of DNA to magnetic beads. DNA is purified in an automated process in which the magnetic beads are transferred through a series of washing buffers. The eluted DNA is suitable for efficient amplification in PCR reactions.

Rapid loop-mediated isothermal amplification assays for grapevine yellows phytoplasmas on crude leaf-vein homogenate has the same performance as qPCR

“…In a final comparative study we prepared samples with the FastPrep-24 (MP Biomedicals) protocol (40 s at 5 m/s), because of its comparably good performance with other protocols (Supplementary Table S1) and its routine use in a DNA extraction protocol (Mehle et al 2013a). We additionally evaluated the analytical sensitivity of direct testing of leaf-vein homogenates prepared with Ultra Turrax Tube Drive device (UTTD, IKA).…”

“…One gram of grapevine plant material (i.e., leaf veins, tendrils, berries, berry pedicels) was added to 2 ml ELISA buffer (Kogovšek et al 2015) in 15 mL test-tubes with garnet matrix and ceramic spheres, and homogenised with FastPrep-24 (MP Biomedicals) for 40 s at 5 m/s. The homogenates then underwent DNA extraction following the procedure described by Mehle et al (2013a). The aliquots of the original homogenates were directly tested without prior DNA extraction.…”

Multilocus sequence typing reveals the presence of three distinct flavescence dorée phytoplasma genetic clusters in Croatian vineyards

“…For the other fraction, the extraction was performed according to Mehle et al . () with the following modifications: 1 g prepared plant tissue was transferred into 15 mL centrifuge tubes containing Matrix A and four 0.25‐inch ceramic spheres (MP Biomedicals). To each tube, 3 mL CTAB buffer (2% CTAB, 1.4 m NaCl, 500 m m EDTA pH 8, 1 m Tris‐HCl pH 8 with freshly added 0.2% 2‐mercaptoethanol) was added.…”

“…Total nucleic acids (TNA) extraction TNA were extracted from 1 g plant material (leaf midribs of grapevine or other plants) according to the previously described CTAB method ( Seruga et al, 2003) for a fraction of the samples. For the other fraction, the extraction was performed according to Mehle et al (2013) with the following modifications: 1 g prepared plant tissue was transferred into 15 mL centrifuge tubes containing Matrix A and four 0.25-inch ceramic spheres (MP Biomedicals). To each tube, 3 mL CTAB buffer (2% CTAB, 1.4 M NaCl, 500 mM EDTA pH 8, 1 M Tris-HCl pH 8 with freshly added 0.2% 2-mercaptoethanol) was added.…”

Test performance study of isothermal amplification tests for the detection of Grapevine flavescence dorée phytoplasma and ‘Candidatus Phytoplasma solani’

“…One gram of leaf mid-vein tissue was homogenized in 2 mL of lysis buffer (from QuickPick TM SML Plant DNA kits; Bio-Nobile) or ELISA buffer (264 mM Tris, 236 mM Tris-HCl, 137 mM NaCl, 2% PVP K-25, 2 mM PEG 6000, 0.05% Tween 20, pH 8.2;Kogov sek et al, 2015), which proved to be as efficient as a lysis buffer in previous trials (Kogov sek et al, 2015), using a tissue homogeniser (FastPrep-24 TM , with a TN 12 9 15-TeenPrep TM Adapter; MP Biochemicals). The total DNA was extracted using QuickPick TM SML Plant DNA kits (Bio-Nobile) and a magnetic particle processor (KingFisher TM mL; Thermo Scientific) (Mehle et al, 2013a). The total DNA extracts were eluted in 200 lL of elution buffer (QuickPick TM SML Plant DNA kit + KingFisher).…”