title: HybProbes-based real-time PCR assay for specific identification of Streptomyces scabies and Streptomyces europaeiscabiei, the potato common scab pathogens.
abstract: The aim of this study was to develop and validate a HybProbes-based real-time PCR assay targeting the trpB gene for specific identification of Streptomyces scabies and Streptomyces europaeiscabiei. Four primer pairs and a fluorescent probe were designed and evaluated for specificity in identifying S. scabies and Streptomyces europaeiscabiei, the potato common scab pathogens. The specificity of the HybProbes-based real-time PCR assay was evaluated using 46 bacterial strains, 23 Streptomyces strains and 23 non-Streptomyces bacterial species. Specific and strong fluorescence signals were detected from all nine strains of S. scabies and Streptomyces europaeiscabiei. No fluorescence signal was detected from 14 strains of other Streptomyces species and all non-Streptomyces strains. The identification was corroborated by the melting curve analysis that was performed immediately after the amplification step. Eight of the nine S. scabies and S. europaeiscabiei strains exhibited a unique melting peak, at Tm of 69·1°C while one strain, Warba-6, had a melt peak at Tm of 65·4°C. This difference in Tm peaks could be attributed to a guanine to cytosine mutation in strain Warba-6 at the region spanning the donor HybProbe. The reported HybProbes assay provides a more specific tool for accurate identification of S. scabies and S. europaeiscabiei strains.
  
  
  CONCLUSIONS
  This study reports a novel assay based on HybProbes chemistry for rapid and accurate identification of the potato common scab pathogens. Since the HybProbes chemistry requires two probes for positive identification, the assay is considered to be more specific than conventional PCR or TaqMan real-time PCR. The developed assay would be a useful tool with great potential in early diagnosis and detection of common scab pathogens of potatoes in infected plants or for surveillance of potatoes grown in soil environment.
authors.affiliation: Agriculture and Agri-Food Canada, Ottawa, ON, Canada.
authors.forename: R
authors.initials: R
authors.lastname: Xu
authors.affiliation: Department of Chemistry, Carleton University, Ottawa, ON, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Falardeau
authors.affiliation: Department of Chemistry, Carleton University, Ottawa, ON, Canada.
authors.forename: T J
authors.initials: TJ
authors.lastname: Avis
authors.affiliation: Agriculture and Agri-Food Canada, Ottawa, ON, Canada.
authors.forename: J T
authors.initials: JT
authors.lastname: Tambong
mesh_terms: D015373:Bacterial Typing Techniques / Q000379:methods*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D011198:Solanum tuberosum / Q000382:microbiology*; D013302:Streptomyces / Q000145:classification* / Q000235:genetics*
chemical_list: D017931:DNA Primers
keywords: Fluorescence Resonance Energy Transfer; HybProbes; Streptomyces; common scab pathogens; identification; potato