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Detection Methods for CGRMV
Molecular Methods
RT-PCR assay developed by W. Jelkmann
RT reaction
- Mix the following components in a 1.5 ml microcentrifuge tube:
- 5 µl purified nucleic acid extract
- 1 µl primer mix 100 pmol/µl hexamer and 0.5 µg/µl
oligo-dT17- primer
- 6 µl RNase-free water
- Incubate at 70° C for 5 min. to denature the RNA
- Place on ice and add the following components:
- 4 µl 5 x MMLV RT buffer
- 2 µl 100 mM DTT
- 1 µl 10 mM dNTP mix
- 1 µl MMLV-RT (200 units)
- Incubate at room temperature for 5 minutes
- Incubate at 37° C for 60 minutes
- Incubate at 70° C for 15 minutes
- Store on ice or at -20° C until required for use in PCR
PCR reaction for CGRMV detection
This reaction will also detect ERMV and some strains of CNRMV.
Reaction mix:
- 5 µl 10 x buffer
(750 mM Tris-HCl pH 9.0, 250 mM (NH4)2SO4,
0.1 % (w/v) Tween 20)
- 5 µl 25 mM MgCl2
- 1 µl 10 mM dNTPs
- 1 µl 10 µM primer mix
- 1 µl cDNA
- 1 µl Taq polymerase (approx. 5 units)
- 36 µl sterile water
Primers:
- GRMV7950: 5´-GCA GCC TTT GAC TTT TTT GAG-3´
- GRMV8316: 5´-CCT ATA GCC AGT CTT CAT ATT ATG-3´
Temperature profile:
94º - 2 min / (94º - 1 min. / 56º - 1 min. / 72º - 1 min.) x 35 /
72º - 5 min.
PCR reaction for combined
CNRMV, CGRMV, ERMV and CNMV detection
Reaction mix:
- 5 µl 10 x Buffer
(100 mM Tris-HCl pH 9.2, 250 mM KCl, 35 mM MgCl2)
- 2 µl foramide
- 1 µl 10 mM dNTPs
- 1 µl 10 µM primer mix
- 1 µl cDNA
- 1 µl Taq polymerase (approx. 5 units)
- 39 µl sterile water
Primers:
- NEG1U: 5´-AGT TCG CAG CYT TTG AYT TYT TTG-3´
- NEG1L: 5´-GAK TGG RWT TGC AGR GGT TTA TCA-3´
Temperature profile:
94º - 2 min / (94º - 1 min. / 55º - 1 min. / 72º - 1 min.) x 35 /
72º - 5 min.
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