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Detection Methods for PD

Molecular Methods

Nested PCR assay developed by M. Heinrich at the IAM

Nucleic acid extraction
DNA is extracted as described by Kobayashi et al. 1998. 100 mg of plant tissue are frozen in liquid nitrogen and homogenized using a bead mill. 1500 µl of Buffer A (50 mM Tris.HCl pH 8.0, 5 mM EDTA, 350 mM Sorbitol, 10 % PEG 8000, 0.1 % Mercaptoethanol) are added and the homogenate centrifuged for 15 min. at 10000 g. The pellet is resuspened in fresh Buffer A and recentrifuged until the supernatant is not viscous anymore. Afterwards the pellet is resuspended in 500 µl Buffer B (50 mM Tris.HCl pH 8.0, 5 mM EDTA, 350 mM Sorbitol, 710 mM NaCl, 1 % Na-Lauroylsarcosin, 0.1 % CTAB, 0.3 % Mercaptoethanol), incubated at 65° C for 15 min. and centrifuged for 5 min. at 10000 g. The supernatant is transferred to a new tube and purified by CI _24:1 extraction. DNA is precipitated with 2/3 vol. i-Propanol at -80° C overnight, centrifuged for 15 min. at 13000 g, washed with 70 % EtOH and resuspended in 100 µl TE. After RNase digestion and subsequent CI extraction DNA is finally precipitated in 2.5 vol. EtOH_abs. and dissolved in a final volume of 50 µl TE. DNA content is determined by UV spectroscopy.

PCR reaction
PCR is carried out in a volume of 20 µl containing 0.5 µl of dNTP (10 mM each), 0.3 µl of each primer (20 pmol/µl), 2 µl 10x PCR buffer (Promega, Madison, WI, USA), 1.4 µl MgCl₂ (25 mM, Promega) 0.1 µl Taq polymerase (5 u/µl, Promega) and 100 ng template DNA.
The primer pair PA2F: 5´-GCC CCG GCT AAC TAT GTG C-3´ and PA2R: 5´-TTG GTG GGC CTA AAT GGA CTC-3´ amplifies a 1187 bp product between nt positions 482 and 1669. After 40 cycles only severe infected plants give a visible band on an agarose gel.
1 µl of a 1:50 dilution of the first PCR product is used as template for nested PCR with the primers NPA2F: 5´-ATG ACC TGG GCT ACA AAC GTG A-3´ and NPA2R: 5´-GGT GGG CCT AAA TGG ACT CG-3´ amplifying a 485 bp product ranging from nt positions 1182 to 1667. The product is loaded to a 2 % agarose gel and detected by UV transillumination.

References

Heinrich M., Botti S., Caprara L., Arthofer W., Strommer S., Hanzer V., Katinger H., Bertaccini A. and Laimer da Câmara Machado M., 2001. Improved detection methods for fruit tree phytoplasmas. Plant Molecular Biology Reporter 19: 169-179.
Kobayashi N., Horikoshi T., Katsuyama H., Handa T. and Takayanagi K., 1998. A simple and efficient DNA extraction method for plants, especially woody plants. Plant Tissue Culture and Biotechnology Vol. 4 No. 2.

last updated October 8, 2001 by Siegfried.Huss