Development of a PCR Assay for Identification of Staphylococci at Genus and Species Levels

Abstract

We have developed a PCR-based assay which allows the detection of staphylococci at the genus level by targeting the tuf gene, which encodes the elongation factor Tu. Degenerate PCR primers derived from consensus regions of several tuf genes were used to amplify a target region of 884 bp from 11 representative staphylococcal species. Subsequently, the entire nucleotide sequence of these amplicons was determined. The analysis of a multiple alignment of these sequences revealed regions conserved among staphylococci but distinct from those of other gram-positive bacteria genetically related to staphylococci. PCR primers complementary to these regions could amplify specifically and efficiently a DNA fragment of 370 bp for all of 27 different staphylococcal species tested. There was no amplification with genomic DNA prepared from 53 nonstaphylococcal species tested to verify the specificity of the assay (20 gram positive and 33 gram negative). Furthermore, this assay amplified efficiently all 27 American Type Culture Collection (ATCC) staphylococcal reference strains as well as 307 clinical isolates of staphylococci from the Québec City region. Analysis of the multiple sequence alignment for the 884-bp fragment for the 11 staphylococcal species as well as comparison of the sequences for the 370-bp amplicon from five unrelated ATCC and clinical strains for each of the species S. aureus, S. epidermidis, S. haemolyticus, S. hominis, and S. saprophyticus demonstrated sufficient interspecies polymorphism to generate genus-and species-specific capture probes. This sequence information allowed the development of Staphylococcus-specific and species-specific (targeting S. aureus, S. epidermidis, S. haemolyticus, S. hominis, or S. saprophyticus) capture probes hybridizing to the 370-bp amplicon. In conclusion, this PCR assay is suitable for detection of staphylococci at both genus and species levels.

Microfluidic Device for Rapid (<15 min) Automated Microarray Hybridization

“…Amplicons of 368 bp were generated either by standard PCR or by asymmetrical PCR amplification of purified staphylococcal genomic DNA using the Staphylococcusspecific primers described previously (24 ). Genomic DNAs were purified from strains S. aureus ATCC 43300, S. epidermidis ATCC 14990, S. haemolyticus ATCC 29970, and S. saprophyticus ATCC 35552, as described previously (24 ).…”

“…Genomic DNAs were purified from strains S. aureus ATCC 43300, S. epidermidis ATCC 14990, S. haemolyticus ATCC 29970, and S. saprophyticus ATCC 35552, as described previously (24 ). Fluorescent Cy dyes were incorporated during asymmetClinical Chemistry 51, No.…”

tuf Gene Sequence Analysis Has Greater Discriminatory Power than 16S rRNA Sequence Analysis in Identification of Clinical Isolates of Coagulase-Negative Staphylococci

“…The tuf gene, which encodes the elongation factor Tu, is an essential constituent of the bacterial genome and is involved in peptide chain formation. Due to its essential nature, it is preferred for diagnostic purposes (19). tuf gene analysis has been shown to be a reliable and reproducible method of identifying CNS; further, it has exhibited better resolution for distinguishing between certain CNS species than 16S rRNA analysis (15,22).…”

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“…To discriminate between staphylococcal species by means of DNA detection and sequencing polymorphisms, 16S rRNA genes are ideal (Becker, K. et al, 2004) but the hsp60 (Goh, S.H. et al, 1997), femA (Vannuffel, P. et al, 1999), sodA (Poyart, C. et al, 2001), tuf (Martineau, F. et al, 2001), rpoA (Drancourt, M. and Raoult, D., 2002), gap (Yugueros, J. et al, 2000).…”