Use of [32P]RNA probes for the dot-hybridization detection of potato spindle tuber viroid.
A dot-hybridization assay using 32P-labelled RNA probes (+RNA and cRNA) transcribed from potato spindle tuber viroid (PSTV) cDNA was described. A complete cDNA copy of PSTV, originally cloned in pBR 322 (pAV 401) was subcloned in the BamHI site of a 'Riboprobe' cloning vectors pSP 64 and pSP 65 in opposite orientations. The reconstructed plasmids were designated pDX 1 and pDX 4, respectively. Transcription of pDX 1 and pDX 4 plasmids by SP6 RNA polymerase resulted in the generation of PSTV-specific RNA (+RNA) and PSTV complementary RNA (cRNA), respectively. The cRNA probe was much more sensitive than the +RNA probe and the nick-translated cDNA probe from the plasmid pAV 401 for the detection of PSTV in clarified plant sap. As little as 1.4 pg of purified PSTV mixed in clarified sap from uninoculated tomato leaves has been detected using cRNA probe. A relatively simple procedure using cetyltrimethyl ammonium bromide (CTAB) as nucleic acid precipitant and an enrichment step for the purification of PSTV was described.
D004262:DNA Restriction Enzymes; D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI; D015247:Deoxyribonuclease HindIII; D009693:Nucleic Acid Hybridization; D010942:Plant Viruses / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids; D012367:RNA, Viral / Q000032:analysis*; D014772:Viroids / Q000235:genetics / Q000302:isolation & purification*
D012367:RNA, Viral; D004262:DNA Restriction Enzymes; D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI; D015247:Deoxyribonuclease HindIII