Development of PCR-based hybridization protocol for identification of streptococcal species.

Journal of Clinical Microbiology
R W Bentley, J A Leigh

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.

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Citations

Dec 14, 2001·Journal of Bacteriology·Philip N Ward, James A Leigh
Jan 1, 1996·Microbiology and Immunology·T IgarashiN Goto
Aug 15, 2003·Journal of Veterinary Medicine. B, Infectious Diseases and Veterinary Public Health·A A HassanC Lammler
Feb 28, 2014·International Journal of Systematic and Evolutionary Microbiology·Ruben Avendaño-HerreraJesús L Romalde
Aug 31, 2002·Journal of Dairy Science·I Meiri-BendekY Kashi
Mar 7, 2003·FEMS Microbiology Letters·A A HassanC Lämmler
Jan 12, 2007·Journal of Clinical Microbiology·Marjo HaanperäKaisu Rantakokko-Jalava
Dec 8, 2005·Journal of Clinical Microbiology·Asa InningsBjörn Herrmann
Feb 19, 2008·Veterinary Microbiology·Jesús L RomaldeAlicia E Toranzo

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