[Design of a universal biotin-containing oligonucleotide diagnostic kit for detecting viroid plant diseases]. / Konstruirovanie universal'nogo biotinsoderzhashchego oligonukleotidnogo diagnostikuma dlia vyiavleniia viroidnykh zabolevanii rastenii.

A non-radioactive diagnosticum for plant viroid diseases has been designed, based on hybridization of a biotin-labeled 26-member oligonucleotide probe with the viroid RNA site identical for potato spindle tuber viroid and chrysanthemum stunt viroid. The biotin label has been introduced into the synthetic oligonucleotide probe by the high-yield acylation of the oligonucleotide aminoalkylamide with the biotin imidazolide or N-hydroxysuccinimide ester. Hybridization techniques have been elaborated for nucleic acids isolated from plant sap. The hybrids obtained have been detected with streptavidin and biotinylated alkaline phosphatase or with the covalent conjugate of streptavidin and alkaline phosphatase, the sensitivity being as low as 1 ng. The methodology used can be applied for revealing viroids and for large scale and quick investigation of plant cell cultures.

[Determination of potato spindle tumor viroid and chrysanthenum stund viroid using biotinylated olideoxyribonucleotides]. / Opredelenie viroidov veretenovidnosti klubnei kartofelia i karlikovosti krizantem s ispol'zovaniem biotinilirovannogo oligodezoksiribonukleotida.

A 26 base long oligodeoxyribonucleotide complementary to a common RNA sequence of potato spindle tuber viroid (PSTV) and chrysantemum stunt viroid (CSV) was synthesized. The 3'-end biotinylated one was used for the detection of PSTV and CSV RNA immobilized on nitrocellulose filters by nucleic acid hybridization. Visualization of hybridization results was performed by two ways, either by streptavidin-alkaline phosphatase conjugate or streptavidine and biotinylated alkaline phosphatase. It was possible to detect 0.65 ng of purified CSV and PSTV RNA. The suggested system of viroid diseases detection can be used by agricultural and horticultural enterprises.

[Cloning of Chrysanthium stund virion cDNA in pUC19 plasmid and the use of cloned cDNA for detecting the virion]. / Klonirovanie kDNK viroida karlikovosti khrizantem v plazmide pUC19 i ispol'zovanie klonirovannoi kDNK dliia obnaruzheniia viroida karlikovosti khrizantem.

26 base long deoxyribonucleotide complementary to the lower part of the Central Conserved Region of chrysanthemum stund viroid (CSV) was used for synthesis of the first strand cDNA. The cDNA was cloned into plasmid vector pUC19 and the primary structure was determined. Cloned, full length cDNA was used as hybridisation probe for detection of CSV. It was possible to detect about 26 pg of purified CSV RNA immobilized on nitrocellulose filters using 32P-labeled probe. In the case of biotinylated probe it was possible to detect about 26 pg of purified CSV RNA visualizing results by streptavidin-alkaline phosphatase conjugates. It has been shown that such a cloned cDNA can be used for wide scale detection of CSV.