Species-specific probes and real-time PCR as a tool for fast detection and differentiation of 15 bacteria relevant in intensive care medicine

Detection of bacterial DNA from laboratory-prepared specimens such as water, urine, and blood has the potential to improve diagnostic tools in microbiology. A novel real-time PCR-based assay was developed and its performance and robustness were evaluated for a panel of spiked suspensions of 15 clinically relevant bacteria. As low as ten colony forming units (CFU)/100 mu l were detectable. No cross-reactivity was observed, except for Staphylococcus aureus and Staphylococcus epidermidis. Nevertheless, S. aureus and S. epidermidis were reliably differentiated by melting curve analysis. The protocol was also validated with three groups containing a mixture of five spiked bacteria each, with the result of reliable differentiation. This novel assay allows an exact identification of 15 microbes relevant in intensive care medicine, including mixed infections, in a one run experiment in less than 4 h.