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Broad-range real-time PCR and sequencing of the 16S rRNA gene region is a widely known method for the detection and identification of bacteria in clinical samples. However, because of the need for sequencing, such identification of bacteria is time-consuming. The aim of our study was to develop a more rapid 16S real-time PCR-based identification assay using species- or genus-specific probes. The Gram-negative bacteria were divided intoPseudomonas species,Pseudomonas aeruginosa ,Escherichia coli , and other Gram-negative species. Within the Gram-positive species, probes were designed forStaphylococcus species,Staphylococcus aureus ,Enterococcus species,Streptococcus species, andStreptococcus pneumoniae . The assay also included a universal probe within the 16S rRNA gene region for the detection of all bacterial DNA. The assay was evaluated with a collection of 248 blood cultures. In this study, the universal probe and the probes targetingPseudomonas spp.,P. aeruginosa ,E. coli ,Streptococcus spp.,S. pneumoniae ,Enterococcus spp., andStaphylococcus spp. all had a sensitivity and specificity of 100%. The probe specific forS. aureus showed eight discrepancies, resulting in a sensitivity of 100% and a specificity of 93%. These data showed high agreement between conventional testing and our novel real-time PCR assay. Furthermore, this assay significantly reduced the time needed for identification. In conclusion, using pathogen-specific probes offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

Molecular Probes for Diagnosis of Clinically Relevant Bacterial Infections in Blood Cultures