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Detection Methods for CNRMV



Molecular Methods

RT-PCR assay developed by W. Jelkmann



RT reaction
  1. 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
  2. Incubate at 70° C for 5 min. to denature the RNA
  3. 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)
  4. Incubate at room temperature for 5 minutes
  5. Incubate at 37° C for 60 minutes
  6. Incubate at 70° C for 15 minutes
  7. Store on ice or at -20° C until required for use in PCR


PCR reaction for CNRMV detection

Under lower stringency this PCR reaction will also detect CGRMV and ERMV.

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:
  • NRM48U: 5´-TTA ATG ATC TTC GTG GCT TGT TG-3´
  • NRM48L: 5´-GAA TTG ACT CCT CGG TGG GTT TA-3´

Temperature profile:

94º - 2 min / (94º - 1 min. / 62º - 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.





last updated October 29, 2001 by Siegfried.Huss