The inner primers were designed with a
digoxigenin or a fluorescein moiety at their 51 end; thus, the short products were labelled with
digoxigenin for the SJNNV genotype or fluorescein for the RGNNV genotype.
The PCR solution and carbon particles conjugated with avidin were directly added to the nitrocellulose membrane with two test lines of immobilized antibodies specific for
digoxigenin and fluorescein.
For labeling of PCR products, the reaction of PCR was performed by dNTP mixture containing
digoxigenin labeling mix (
Digoxigenin dNTPs, Roche, Germany) with the same condition.
The synthesis of the
digoxigenin labeled riboprobe and the hybridization reactions with the polyprobe were conducted as previously described (HERRANZ et al., 2005) with some modifications.
The prepared
digoxigenin labeledcDNA probe was used in dot blot and tissue print hybridization assays.
The
digoxigenin tail-labelled PGRS (5' CGG CCG TTG CCG CCG TTG CCG CCG TTG CCG CCG 3') or DR (5' CCG AGA GGG GAC GGA AAC 3') probes were used for hybridization which was performed at 65[degrees] C using rolling bottle method (Table 2).
Digoxigenin labelled probes were used at a concentration of 80 ng/100 [micro]L in hybridization buffer [Tris-HCl 0.02 M pH 7,5; NaCl 0.3 M; EDTA 0.01 M; DTT 0.1 M; formamide 50%; Denhardt's 1X; tRNA 100 [micro]g/mL; ss-DNA 100 [micro]g/mL] overnight at 50[degrees]C in a moist chamber.
Sections were incubated in a
digoxigenin antibody solution (500:1 dilution) containing 2.5 mL buffer 1 (100 mM Tris, 150 mM NaCl, 0.3% Triton X-100, 1% goat serum, pH 7.5) with 5 [micro]L of antidigoxigenin Fab fragments conjugated with alkaline phosphatase (750 U/mL) (Roche, Mannheim, Germany).
Some of the useful plant drugs include vinblastine, vincristine, Taxol, Podophyllotoxin, camptothecin, digitoxigenin, gitoxigenin,
digoxigenin, tubocurarine, morphine, codeine, aspirin, atropine, Pilocarpine, capsicum, Ellison, curcumin, artemesinin and ephedrine among others.