Highly sensitive digoxigenin-labelled DNA probe for the detection of potato spindle tuber viroid.
A molecular probe pSPAv6.2(+), with concatameric insert representing 6.2-times repeated copy of potato spindle tuber viroid (PSTV) RNA, was labelled with digoxigenin and used to detect PSTV by dot-blot hybridization assay. The probe was highly sensitive and specific, detecting as little as 2.5 pg of PSTV RNA. Both severe and mild PSTV strains were detectable in 64-512-times diluted crude extracts from infected tomato leaves, and potato leaves, sprouts, and seeds. For extraction of plant tissue three buffers were compared to determine the lowest non-specific background and the highest sensitivity. The results showed that the digoxigenin-labelled probe is as sensitive as the 32P-labelled probe and can replace radioactive techniques in PSTV detection. With such high sensitivity, the probe is also potentially useful for detecting the viroid in composite samples of mass-indexing programs.
Welnicki
M
M
Department of Plant Science, University of Alberta, Edmonton, Canada.
Hiruki
C
C
D015342:DNA Probes; D004076:Digoxigenin; D005069:Evaluation Studies as Topic; D005638:Fruit / Q000382:microbiology; D015336:Molecular Probe Techniques / Q000706:statistics & numerical data*; D009693:Nucleic Acid Hybridization; D010942:Plant Viruses / Q000235:genetics* / Q000302:isolation & purification; D012367:RNA, Viral / Q000235:genetics; D012680:Sensitivity and Specificity; D011198:Solanum tuberosum / Q000382:microbiology; D014773:Virology / Q000379:methods* / Q000706:statistics & numerical data
D015342:DNA Probes; D012367:RNA, Viral; D004076:Digoxigenin