A mixture of synthetic oligonucleotide probes labeled with biotin for the sensitive detection of potato spindle tuber viroid.
Five kinds of synthetic oligonucleotide probes labeled with biotin (BIO) were designed for the detection of potato spindle tuber viroid (PSTVd), and their sensitivities were compared with that of a digoxigenin (DIG)- or BIO-labeled cDNA probe. Although each oligonucleotide probe alone was less sensitive than the DIG-cDNA probe, sensitivity was increased by using a mixture of two or all of the five oligonucleotide probes. The sensitivity of a PSmix1-5 probe, which was a mixture of five oligonucleotides, was the same as that of a DIG-labeled cDNA probe, which can detect 7.8 pg of purified PSTVd and PSTVd in nucleic acids, equivalent to extracts from 20 microg of infected potato leaf and 310 microg of infected potato tuber. Using the PSmix1-5 probe, PSTVd in all leaves and tubers of seven potato cultivars could be detected without any background. Moreover, with the PSmix1-5 probe, the hybridization time could be shortened to 2 h without any decrease in sensitivity, whereas the sensitivity of the cDNA probes clearly decreased when the hybridization time was shortened. Hybridization using a mixture of several oligonucleotide probes may be applicable to the gene diagnosis of other viroids and viruses.
Nakahara
K
K
Department of Agrobiology and Bioresources, Faculty of Agriculture, Hokkaido University, Sapporo, Japan.
Hataya
T
T
Hayashi
Y
Y
Sugimoto
T
T
Kimura
I
I
Shikata
E
E
D001483:Base Sequence; D001710:Biotin; D015342:DNA Probes; D018076:DNA, Complementary; D004076:Digoxigenin; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010942:Plant Viruses / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D011198:Solanum tuberosum / Q000821:virology*; D014772:Viroids / Q000235:genetics* / Q000302:isolation & purification*
D015342:DNA Probes; D018076:DNA, Complementary; D015345:Oligonucleotide Probes; D001710:Biotin; D004076:Digoxigenin