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Detection Methods for AP

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.
Sequence alignment using the GenBank Higher Plant sequence database showed no significant homology between the primer sequences and chloroplast sequences, plastid 16S sequences or to any published plant sequences. Database searches in the NCBI Blast Search showed full homology to 16 phytoplasma sequences.

Phytoplasma detection in microgropagated shootes, developed by A. Bertaccini

Nucleic acid extraction by silica-captue

Grind 200 - 500 mg sample in 1 ml PBS-Tween buffer supplemented with 2 % (w/v) PVP (PVP K25, Fluka) and 20 mM DIECA
Spin for 10 min. at 13000 rpm / 4° C in an Eppendorf type centrifuge
Transfer 200 µl of supernatant to new tube
Add 20 µl 10 % (w/v) SDS solution
Incubate 15 min. at 55° C
Add 100 µl of 3 M potassium acetate, mix well
Incubate 5 min. on ice
Spin 5 min. as above, trasfer supernatant to a new tube
Add 700 µl of 6 M NaI solution, mix well
Add 5 µl of well resuspended silicagel solution (see below), mix well
Incubate 10 min. at room temperature
Spin for 1 min. at 5000 rpm
Discard supernatant, add 500 µl washing solution (10 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 50 mM NaCl, 50 % (w/v) Ethanol)
Resuspend silica pellet and repeat washing step once
Dry pellet completely and resuspend it in 400 µl sterile water
Incubate 5 min. at 55° C
Spin 2 min. at 13000 rpm; the supernatant is ready to be used in PCR

Preparation of silicagel solution

Add 60 g silica particles (Sigma S5631) to 500 ml ultrapure water in a measuring cylinder, mix well
Let the suspension settle for 24 hours at room temperature
Discard 470 ml of the supernatant
Refill with ultrapure water to a final volume of 500 ml, mix well
Let settle for 5 hours, discard 440 of the spernatant
Adjust the pH of the remaining slurry to 2.0 using concentrated HCl
Divide in small aliquots (approx. 5 ml) and autoclave for 20 min. at 120° C
Store in the dark at room temperature

PCR procedure
The PCR mastermix contains of 12.5 µl DNA extract, 5.375 µl water, 2.5 µl 10x PCR buffer, 250 µM dNTPs, 0.3 µM each forward and reverse primer and 0.16 U Taq. If a nested PCR follows, 1 µl of the first PCR product, 17.875 µl water, 2.5 µl 10x PCR buffer, 250 µM dNTPs, 0.3 µM each forward and reverse primer and 0.16 U Taq are used.

Primer pair for direct PCR
P1 (Deng et Hiruki, 1991) 5´-AAG AAT TTG ATC CTG GCT CAG GAT T-3´
m23SR1832r (Schneider et al., 1995) 5´-CGT CCT TCA TCG GCT CTT-3´

Primer pairs for nested PCR
R16F2 (Lee et al., 1995) 5´-GAA ACG ACT GCT AAG ACT GG-3´
R16R2 (Lee et al., 1995) 5´-TGA CGG GCG GTG TGT ACA AAC CCC G-3´

R16(X)F1₁₉₇ (Lee et al., 1994) 5´-GAC CCG CAA GTA TGC TGA GAG ATG-3´
R16(X)R1₁₂₉₇ (Lee et al., 1994) 5´-CAA TCC GAA CTG AGA CTG T-3´

R16M1₇₅₈ (Gibb et al., 1995) 5´-GTC TTT ACT GAC GC-3´
R16M2₁₂₃₂ (Gibb et al., 1995) 5´-CTT CAG CTA CCC TTT GTA AC-3´

Temperature profile for all direct and nested primers
2 min./94° - (1 min./94° - 2 min./50° - 3 min/72°) x 35 - 10 min./72°

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