Use of tuf sequences for genus-specific PCR detection and phylogenetic analysis of 28 streptococcal species - PubMed (original) (raw)

Use of tuf sequences for genus-specific PCR detection and phylogenetic analysis of 28 streptococcal species

François J Picard et al. J Clin Microbiol. 2004 Aug.

Abstract

A 761-bp portion of the tuf gene (encoding the elongation factor Tu) from 28 clinically relevant streptococcal species was obtained by sequencing amplicons generated using broad-range PCR primers. These tuf sequences were used to select Streptococcus-specific PCR primers and to perform phylogenetic analysis. The specificity of the PCR assay was verified using 102 different bacterial species, including the 28 streptococcal species. Genomic DNA purified from all streptococcal species was efficiently detected, whereas there was no amplification with DNA from 72 of the 74 nonstreptococcal bacterial species tested. There was cross-amplification with DNAs from Enterococcus durans and Lactococcus lactis. However, the 15 to 31% nucleotide sequence divergence in the 761-bp tuf portion of these two species compared to any streptococcal tuf sequence provides ample sequence divergence to allow the development of internal probes specific to streptococci. The Streptococcus-specific assay was highly sensitive for all 28 streptococcal species tested (i.e., detection limit of 1 to 10 genome copies per PCR). The tuf sequence data was also used to perform extensive phylogenetic analysis, which was generally in agreement with phylogeny determined on the basis of 16S rRNA gene data. However, the tuf gene provided a better discrimination at the streptococcal species level that should be particularly useful for the identification of very closely related species. In conclusion, tuf appears more suitable than the 16S ribosomal RNA gene for the development of diagnostic assays for the detection and identification of streptococcal species because of its higher level of species-specific genetic divergence.

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Figures

FIG. 1.

Phylogenetic relationships among 28 streptococcal species. (A) Phylogenetic tree based on a 761-bp portion of tuf. (B) Phylogenetic tree based on a 1,260-bp portion of 16S rDNA. The trees were generated using the MEGA2 heuristic method, and evolutionary distance values were calculated by Kimura's two-parameter substitution model. The value on each branch represents the percentage of bootstrap replications supporting the branch. A total of 1,000 bootstrap replications were calculated. Bootstrap values lower than 50% are not shown. GenBank accession numbers are given in parentheses. The tuf and 16S rDNA portions correspond to nucleotide positions 340 to 1,100 of the complete tuf gene of S. pneumoniae R6 (AE008504) and 93 to 1,382 of the complete 16S rRNA gene of S. pneumoniae R6 (AE008546). All sequences used for these phylogenetic analysis were obtained either from this study or from the following sources: GenBank (

http://www.ncbi.nlm.nih.gov

) (A), TIGR ongoing genome projects (

http://www.tigr.org

) (B), Sanger ongoing genome projects (

http://www.sanger.ac.uk

) (C), and our group (for previously determined sequences) (24) (D); sequences from these four sources are indicated with A, B, C, and D, respectively.