Development of a PCR assay for identification of staphylococci at genus and species levels

We have developed a PCR-based assay which allows the detection of staphyloc occi at the genus level by targeting the tuf gene, which encodes the elonga tion factor Tu. Degenerate PCR primers derived from consensus regions of se veral tuf genes were used to amplify a target region of 884 bp from 11 repr esentative staphylococcal species. Subsequently, the entire nucleotide sequ ence of these amplicons was determined. The analysis of a multiple alignmen t of these sequences revealed regions conserved among staphylococci but dis tinct from those of other gram-positive bacteria genetically related to sta phylococci. PCR primers complementary to these regions could amplify specif ically and efficiently a DNA fragment of 370 bp for all of 27 different sta phylococcal species tested, There was no amplification with genomic DNA pre pared 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 (P;TCC) stap hylococcal reference strains as well as 307 clinical isolates of staphyloco cci from the Quebec City region, Analysis of the multiple sequence alignmen t for the 884-bp fragment for the 11 staphylococcal species as well as comp arison of the sequences for the 370-bp amplicon from five unrelated ATCC an d clinical strains for each of the species S. aureus, S. epidermidis, S. ha emolyticus, S. hominis, and S. saprophyticus demonstrated sufficient inters pecies polymorphism to generate genus- and species-specific capture probes. This sequence information allowed the development of Staphylococus-specifi c and species-specific (targeting S. aureus, S. epidermidis, S. haemolyticu s, S. hominis, or S. saprophyticus) capture probes hybridizing to the 370-b p amplicon. In conclusion, this PCR assay is suitable for detection of stap hylococci at both genus and species levels.