Detection of Chrysanthemum stunt viroid (CSV) using nick translated probes in a dot-blot hybridization assay.
We developed a dot-blot hydridization assay for the detection of Chrysanthemum stunt viroid (CSV) in Chrysanthemum plant samples. The probe, a recombinant plasmid containing a full-length monomeric cDNA copy of CSV, is labelled with (32P) by nick-translation. The influence of the hybridization conditions, of the sample denaturation technique and of the plant sap components on the final sensitivity has been studied. The optimized system, involving a formaldehyde denaturation step, allows the detection of as little as 5 pg of purified viroid. Under these conditions, 100 pg of pure viroid diluted in plant sap, or infected plant extract diluted 1:25 in healthy extract can be detected, showing the potential of this method for indexing of Chrysanthemum for CSV infection.
Candresse
T
T
Station de pathologie végétale, INRA, Centre de Recherches de Bordeaux, Pont de la Maye, France.
Macquaire
G
G
Monsion
M
M
Dunez
J
J
D011723:Chrysanthemum cinerariifolium / Q000382:microbiology*; D004274:DNA, Recombinant; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D010935:Plant Diseases; D010942:Plant Viruses / Q000302:isolation & purification*; D010944:Plants / Q000382:microbiology*; D014772:Viroids / Q000302:isolation & purification*
D004274:DNA, Recombinant