|
German Version
Site map
FAIR 3889
Virus diseases
Phytoplasma diseases
Pathogen collection
Pathogen detection
ACLSV
ApMV
ASGV
ASPV
PPV
PDV
PNRSV
ArMV
ToRSV
RpRSV
SLRSV
GFLV
GLRaV-1
GLRaV-3
LChV
CMLV
CRMV
CNRMV
CGRMV
ChTLV
CVA
AP
ESFY
PD
Pathogen elimination
Contact us
Related Sites
|
Detection Methods for ApMV
Serological Methods
ApMV 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
PCR-ELISA for simultaneous detection of ApMV and PNRSV
developed by T. Candresse
Sample preparation
Grind plant samples 1:10 (w/v) in a PBS-Tween-Buffer supplemented
with 2 % polyvinylpyrrolidone K25 and 20 mM sodium diethydithiocarbamate.
Clarify extract by centrifugation (10 min., 13000 rpm, 4° C).
Coating
Coat Eppendorf tubes with 100 µl of a 2 µg/ml solution
of anti-PNRSV-IgG, anti-ApMV-IgG or a 1:1 mixture of both
antibodies. After coating, wash the Eppendorf tubes with PBS-Tween
buffer and add 100 µl of the plant extract, incubate 3 h at
37° C, wash again with PBS-Tween buffer and allow to dry.
RT-PCR-Assay
Add 50 µl of PCR mix (10 mM Tris.HCl, pH 8.8, 1.5 mM
MgCl2, 50 mM KCl, 0.3% Triton X100, 250 µM dNTPs,
1 µM of each primer, 0.5 U AMV reverse transcriptase, 1 U
Taq polymerase) to each eppi.
Primer ILAR1: 5'-TTC TAG CAG GTC TTC ATC GA-3'
Primer ILAR2: 5'-CAA CCG AGA GGT TGG CA-3'
cycling sheme: 15 min. 42°C - 5 min. 92°C -
(20 sec. 92°C - 20 sec. 52 °C - 40 sec. 72°C) x 40
An amplicon of 206 bp can bedetected on a 1 % agarose gel by
electrophoresis and ethidium bromide staining.
|
