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FAIR 3889
Virus diseases
Phytoplasma diseases
Pathogen collection
Pathogen detection
ACLSV
ApMV
ASGV
ASPV
PPV
PDV
PNRSV
PNRSV
ArMV
ToRSV
RpRSV
SLRSV
GFLV
GLRaV-1
GLRaV-3
LChV
CMLV
CRMV
CNRMV
CGRMV
ChTLV
CVA
AP
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Pathogen elimination
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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.
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