title: Amplification of RNA by NASBA allows direct detection of viable cells of Ralstonia solanacearum in potato.
abstract: OBJECTIVE
  The objective of this study was to develop a Nucleic Acid Sequence Based Amplification (NASBA) assay, targeting 16S rRNA sequences, for direct detection of viable cells of Ralstonia solanacearum, the causal organism of bacterial wilt. The presence of intact 16S rRNA is considered to be a useful indicator for viability, as a rapid degradation of this target molecule is found upon cell death.
  
  
  RESULTS
  It was demonstrated by RNase treatment of extracted nucleic acids from R. solanacearum cell suspensions that NASBA exclusively detected RNA and not DNA. The ability of NASBA to assess viability was demonstrated in two sets of experiments. In the first experiment, viable and chlorine-killed cells of R. solanacearum were added to a potato tuber extract and tested in NASBA and PCR. In NASBA, only extracts spiked with viable cells resulted in a specific signal after Northern blot analysis, whereas in PCR, targeting 16S rDNA sequences, both extracts with viable and killed cells resulted in specific signals. In the second experiment, the survival of R. solanacearum on metal strips was studied using NASBA, PCR-amplification and dilution plating on the semiselective medium SMSA. A positive correlation was found between NASBA and dilution plating detecting culturable cells, whereas PCR-amplification resulted in positive reactions also long after cells were dead. The detection level of NASBA for R. solanacearum added to potato tuber extracts was determined at 104 cfu per ml of extract, equivalent to 100 cfu per reaction. With purified RNA a detection level of 104 rRNA molecules was found. This corresponds with less than one bacterial cell, assuming that a metabolically active cell contains ca 105 copies of rRNA. Preliminary experiments demonstrated the potential of NASBA to detect R. solanacearum in naturally infected potato tuber extracts.
  
  
  CONCLUSIONS
  NASBA specifically amplifies RNA from viable cells of R. solanacearum even present in complex substrates at a level of 100 cfu per reaction.
  
  
  CONCLUSIONS
  The novel NASBA assay will be particularly valuable for detection of R. solanacearum in ecological studies in which specifically viable cells should be determined.
authors.affiliation: Plant Research International, Wageningen, The Netherlands.
authors.forename: L
authors.initials: L
authors.lastname: Bentsink
authors.forename: G O M
authors.initials: GO
authors.lastname: Leone
authors.forename: J R C M
authors.initials: JR
authors.lastname: van Beckhoven
authors.forename: H B
authors.initials: HB
authors.lastname: van Schijndel
authors.forename: B
authors.initials: B
authors.lastname: van Gemen
authors.forename: J M
authors.initials: JM
authors.lastname: van der Wolf
mesh_terms: D001483:Base Sequence; D020562:Betaproteobacteria / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification*; D003470:Culture Media; D004275:DNA, Ribosomal / Q000032:analysis / Q000235:genetics; D020459:Genes, rRNA / Q000235:genetics; D007501:Iron / Q000378:metabolism; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000032:analysis* / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000032:analysis / Q000235:genetics; D021161:Self-Sustained Sequence Replication / Q000379:methods*; D017422:Sequence Analysis, DNA; D011198:Solanum tuberosum / Q000382:microbiology*
chemical_list: D003470:Culture Media; D004275:DNA, Ribosomal; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D007501:Iron