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Journal of Clinical Microbiology, August 2004, p. 3686-3695, Vol. 42, No. 8
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.8.3686-3695.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Use of tuf Sequences for Genus-Specific PCR Detection and Phylogenetic Analysis of 28 Streptococcal Species

François J. Picard,1 Danbing Ke,1,2,{dagger} Dominique K. Boudreau,1 Maurice Boissinot,1,2 Ann Huletsky,1,2 Dave Richard,1,2 Marc Ouellette,1,2 Paul H. Roy,1,3 and Michel G. Bergeron1,2*

Centre de Recherche en Infectiologie de l'Université Laval, Centre Hospitalier Universitaire de Québec (Pavillon CHUL), Sainte-Foy, Québec, Canada G1V 4G2,1 Division de Microbiologie, Faculté de Medicine,2 Département de Biochimie et de Microbiologie, Faculté des Sciences et de Génie, Université Laval, Sainte-Foy, Québec, Canada G1K 7P43

Received 6 October 2003/ Returned for modification 15 January 2004/ Accepted 22 April 2004

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.

* Corresponding author. Mailing address: Centre de Recherche en Infectiologie de l'Université Laval, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, 2705 Boul. Laurier, Ste-Foy, Québec, Canada G1V 4G2. Phone: (418) 654-2705. Fax: (418) 654-2715. E-mail: Michel.G.Bergeron{at}crchul.ulaval.ca.

{dagger} Present address: Bioniche Therapeutics Research Center, Montreal, Quebec, Canada H4P 2R2.

Journal of Clinical Microbiology, August 2004, p. 3686-3695, Vol. 42, No. 8
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.8.3686-3695.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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