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FAIR 3889

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Detection Methods for ASGV



Serological Methods

ASGV can be detected by DAS-ELISA. Antibodies and IgG-conjugate are commercially vailable from different sources. The IAM currently uses Bioreba and Loewe diagnosis kits.


Molecular Methods

Immunocapture PCR

The immunocapture procedure follows a protocol by G. Nolasco, UCTA, Faro, Portugal, modified by W. Arthofer, IAM. The primers were developed by J. Kummert in the frame of FAIR 3889.


Sample preparation
150 mg of plant material were ground in 2 ml Eppendorf tubes using a bead mill. 1.5 ml extraction buffer (1x PBS supplemented with 2% PVP and 0.05% Tween 20) were added, votexed and centrifuged for 7 min. at 7000 rpm and 4° C.

Coating and Immunocapture
Eppendorf 96-well PCR plates were coated 3 h at 37° C with 25 µl of a 1:200 dilution of Loewe anti-ASGV-IgG in carbonate buffer, followed by 3 washes with PBS buffer.
50 µl of centrifuged plant extract were added and incubated 3 h at 37° C. Afterwards plates were washed 3 times with PBS buffer.

RT-PCR-Assay
Each well was supplemented with 25 µl of the following reacion mix:
  • 15.45 µl dH2O
  • 2.5 µl Promega 10x Mg-Free PCR Buffer
  • 4.5 µl Promega MgCl2 25 mM
  • 1 µl DTT 100 mM
  • 0.5 µl dNTPs 10 mM
  • 0.3 µl of each primer, 20 µM
  • 0.2 µl MBI RNase inhibitor
  • 0.15 µl MBI MMLV reverse transcriptase, 30 u/µl
  • 0.1 µl Promega Taq polymerase
ASGV-5F and ASGV-5R wre used as primers. The sequences of these primers were developed by J. Kummert and are not published.
Reverse transcription was carried out at 38° C for 45 min. followed by 38 cycles 92° C 30 sec. - 55° C 30 sec. - 72° C 30 sec.
PCR products were analyzed by agarose gel electrophoresis and EtBr staining.


last updated December 20, 2001 by Siegfried.Huss