Study of the intra- and interlaboratory reproducibility of partial single base C-sequencing of the 16S rRNA gene and its applicability for the identification of members of the genus Streptococcus.
The use of Single Base C-Sequencing of the first 500 bases of the 16S rRNA-gene (SBCS) combined with capillary electrophoresis was evaluated for the identification of reference strains of 30 different species within the genus Streptococcus. For SBCS, only dd-CTP's are used in the sequencing reactions instead of the four dideoxy bases and the primer is fluorescently labeled. The reproducibility, interlaboratory exchangeability and discriminative power of this method were studied by comparing the patterns obtained in three laboratories under highly standardized conditions. The interlaboratory reproducibility proved to be high, enabling the construction of a common database for the identification of strains belonging to the streptococcal species studied. Most of the examined species generated distinguishable profiles. SBCS did not differentiate between the closely related species S. constellatus and S. intermedius. Also S. thermophilus and S. vestibularis as well as S. mitis and S. pneumoniae showed highly resembling profiles. The previously reported heterogeneity within the species S. equinus was reflected by SBCS. For all other species, strains belonging to the same species generated indistinguishable patterns. In conclusion, Single Base C-sequencing of the first 500 bases of the 16S rRNA-gene could be a useful and widely applicable method for the identification of bacteria at the species level, with the added advantage of being more rapid and easier to automatize than full sequence determination.
Storms
Virginie
V
Department of Biochemistry, Physiology and Microbiology, Faculty of Sciences, University of Ghent, Gent, Belgium. virginie.storms@rug.ac.be
Baele
Margo
M
Coopman
Renata
R
Willems
Anne
A
de Baere
Thierry
T
Haesebrouck
Freddy
F
Verschraegen
Gerda
G
Gillis
Monique
M
Vaneechoutte
Mario
M
D001483:Base Sequence; D016172:DNA Fingerprinting / Q000592:standards; D004269:DNA, Bacterial / Q000235:genetics; D021901:DNA, Intergenic / Q000235:genetics; D016208:Databases, Factual / Q000706:statistics & numerical data; D019075:Electrophoresis, Capillary / Q000379:methods / Q000592:standards / Q000706:statistics & numerical data; D005798:Genes, Bacterial; D020459:Genes, rRNA; D007753:Laboratories / Q000592:standards; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards* / Q000706:statistics & numerical data; D012336:RNA, Ribosomal, 16S / Q000032:analysis* / Q000145:classification; D015203:Reproducibility of Results; D013290:Streptococcal Infections / Q000175:diagnosis; D013291:Streptococcus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*
D004269:DNA, Bacterial; D021901:DNA, Intergenic; D012336:RNA, Ribosomal, 16S