Detection and differentiation of in vitro-spiked bacteria by real-time PCR and melting-curve analysis

We introduce a consensus real-time PCR protocol for the detection of bacterial DNA from laboratory-prepared specimens such as water, urine, and plasma. This prototype detection system enables an exact Gram stain classification and, in particular, screening for specific species of 17 intensive care unit-relevant bacteria by means of fluorescence hybridization probes and melting-curve analysis in a one-run experiment. One strain of every species was tested at a final density of 10(6) CFU/ml. All bacteria examined except Staphylococcus aureus and Staphylococcus epidermidis could be differentiated successfully; S. aureus and S. epidermidis could only be classified as Staphylococcus species. The hands-on time for preparation of the DNA, performance of the PCR, and evaluation of the PCR results was less than 4 h. Nevertheless, this prototype detection system requires more clinical validation.