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. 2003 Aug;41(8):3481-6.
doi: 10.1128/jcm.41.8.3481-3486.2003.

Identification of Streptococcus Sanguinis With a PCR-generated Species-Specific DNA Probe

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Free PMC article

Identification of Streptococcus Sanguinis With a PCR-generated Species-Specific DNA Probe

Yihong Li et al. J Clin Microbiol. .
Free PMC article

Abstract

The objective of the present study was to design a PCR-generated DNA probe and determine the specificity of the probe for the identification of clinical isolates of Streptococcus sanguinis. To do this, we examined over 200 arbitrarily primed PCR (AP-PCR) amplicon patterns obtained with DNA from clinical isolates of S. sanguinis. A 1.6-kb DNA amplicon that was common to all AP-PCR profiles was extracted from agarose gels and then cloned and sequenced. A search for a similar sequence in the GenBank database with the BLASTN program revealed that the 1.6-kb DNA fragment comprised an intergenic region between two housekeeping genes, uncC (proton-translocating ATPase) and murA (UDP-N-acetylglucosamine enolpyruvyl transferase). Three digoxigenin-labeled DNA probes were synthesized on the basis of the sequence of the 1.6-kb fragment: the sequence of probe SSA-1 contained the proton-translocating ATPase (uncC) and the entire intergenic region, the sequence of probe SSA-2 contained only the intergenic region, and the sequence of probe SSA-3 contained an internal region of the murA gene. Dot blot hybridization showed that the three probes displayed signals for hybridization to both S. sanguinis strain ATCC 10556 and the S. sanguinis clinical isolates. Probe SSA-1, however, hybridized to DNA from S. oralis and S. mitis. Probe SSA-3 hybridized to DNA from S. gordonii, S. mitis, S. oralis, S. parasanguinis, and S. vestibularis. The probe SSA-2-specific intergenic region appeared to be specific for S. sanguinis. The results from this study suggest that probe SSA-2 may serve as a species-specific DNA probe for the identification of clinical isolates of S. sanguinis.

Figures

FIG. 1.
FIG. 1.
AP-PCR fingerprint profiles generated from S. sanguinis, other oral mitis group species, and S. sanguinis clinical isolates. AP-PCR results were obtained by amplification of genomic DNA with primer OPA-02. A 1.6-kb amplicon was observed for all S. sanguinis strains. Other reference strains of the mitis group showed different AP-PCR patterns and the absence of a 1.6-kb amplicon. The non-S. sanguinis strains tested were as follows: 10557, S. oralis; 10558, S. gordonii; 15911, S. parasanguinis; 9811, S. oralis; 903, S. mitis; 49999, S. cristatus; WU2, S. pneumoniae; 35037, S. oralis.
FIG. 2.
FIG. 2.
Locus and composition of DNA-based probes used for dot hybridization. The three probes were designed on the basis of the sequence of a 1,653-bp fragment from strains of S. sanguinis. The sequence of probe SSA-1 comprised a portion of the first ORF (uncC gene) and the entire intergenic region, the sequence of probe SSA-2 comprised only the intergenic region, and the sequence of probe SSA-3 comprised an internal region of the second ORF (murA gene).
FIG. 3.
FIG. 3.
Dot blot hybridization shows the specificities of the three probes hybridized with different Streptococcus reference strains as well as with S. sanguinis clinical isolates. Probe SSA-2 was specific for type strain ATCC 10556 (B) and all S. sanguinis clinical isolates (D). SSA-1 and SSA-3 showed different degrees of hybridization to other species (A and C). The bacterial strains tested were as follows: 1, S. sanguinis; 2, S. oralis; 3, S. gordonii; 4, S. cristatus; 5, S. oralis; 6, S. mitis; 7, S. parasanguinis; 8, S. pneumoniae; 9, S. mutans; 10, S. salivarius; 11, S. sobrinus; 12, S. ratti; 13, S. vestibularis; 14, A. naeslundii; 15, L. acidophilus; 16, E. coli JM109.
FIG. 4.
FIG. 4.
Specific amplification of the 475-bp intergenic region from S. sanguinis with primers F2 and R2. The agarose gel shows the presence of the PCR amplicon for ATCC 10556 and the S. sanguinis clinical isolates and the absence of the PCR amplicon for the other non-S. sanguinis strains tested.

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