title: Development of PCR-based hybridization protocol for identification of streptococcal species.
abstract: 16S rRNA of Streptococcus agalactiae, S. uberis, and S. parauberis was bound to streptavidin-coated magnetic beads by using a biotinylated oligonucleotide probe complementary to a highly conserved region of the molecule. In-solution hybridization of radiolabelled oligonucleotide probes to immobilized 16S rRNA allowed the specific identification of S. agalactiae and S. parauberis but not S. uberis. PCR was used to amplify a species-specific region of the 16S rRNA gene from these species. One of the PCR primers was biotinylated at the 5' end to allow purification of the amplified product on streptavidin-coated magnetic beads and subsequent denaturation to yield immobilized single-stranded DNA. Radiolabelled oligonucleotide probes were hybridized in solution to the single-stranded target molecule and enabled species-specific identification of the target organism. This protocol overcame problems associated with hybridization of the S. uberis-specific probe to 16S rRNA in solution. A similar procedure may enable the specific detection of other streptococci which exhibit a species-specific sequence in this region of the gene.
authors.affiliation: AFRC Institute for Animal Health, Compton, Newbury, United Kingdom.
authors.forename: R W
authors.initials: RW
authors.lastname: Bentley
authors.forename: J A
authors.initials: JA
authors.lastname: Leigh
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D005069:Evaluation Studies as Topic; D005260:Female; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D013291:Streptococcus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D013292:Streptococcus agalactiae / Q000235:genetics / Q000302:isolation & purification
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S