PCR Detection of Gaeumannomyces graminis var avenae
The following protocol is based on the work by Rachdawong, Sansanalak et al, 2002.
The PCR technique employs a common primer (AV3rc) for detection of G. graminis var.
avenae, G. graminis var. tritici, and G. graminis var. graminis. Specificity for
Gaeumannomyces graminis var. avenae is conferred through the use of the Gga-specific
primer (Gga). The accuracy of this assay was determined by sequencing the amplicon
from Gga suspect roots used as positive control.
Sample preparation
Freeze select grass roots in liquid nitrogen placed in MiniBeadBeater tubes with two 6mm glass beads. Beat at 2500 rpm for 30 sec three times, refreezing between each rep.
Extract DNA from pulverized roots using the DNEasy Plant Mini Kit.
PCR Reaction Mixture (per 50 µl reaction)
Nanopure water
FailSafe 2X PCR PreMix E*
50X Titanium Taq DNA polymerase
Primer Gga, 5 µM
Primer AV3rc, 5 µM
18.5 µl
25 µl
0.5 µl
2.5 µl
2.5 µl
Sample
1.0µl of DNA extract (or controls as noted below)
Primers
Prepare to a concentration of 5 µM (5 pmol/µl) in 1X TNE buffer:
Gga
5’- - ACGGCGGTGGATGGCAAGAC-3’
AV3rc
5’- TGCTCATGGTGGTTCCTGCG-3‘
Controls
Negative control:
Positive control #1:
Positive control #2:
1 µl Nanopure water
1 µl genomic DNA from a known Gga DNA sample
1 l of genomic DNA from a known Gga DNA sample plus 1 l
of unknown sample. If results show evidence of PCR inhibition,
retest sample extract at 10-fold dilution and 100-fold dilution.
Cycling Protocol
Name of Protocol:
On GeneAmp 9700: Gga (use max ramp rate) listed under Diagnostics
1. 95ºC for 5 min
2. 35 cycles of: 94ºC for 30 sec
68ºC for 30 sec
3. 72ºC for 10 min
Duration of protocol: 1 h, 10 min
Expected Results
A single product of approx. 617 bp is amplified with these primers.
References
Rachdawong, Sansanalak et al. Plant Disease vol. 86, No 6, 2002, pp 652-660.
*Epicentre Biotechnologies www.EpiBio.com. Pre-mixes F, H, and I are also suitable,
though may yield a slightly more diffuse band on the gel.
Document Control: February 12, 2016