Detection and Differentiation of In Vitro-Spiked Bacteria by Real-Time PCR and Melting-Curve Analysis

Abstract

We introduce a consensus real-time PCR protocol for the detection of bacterial DNA from laboratoryprepared 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.The early detection and adequate treatment of bacterial infections are critical for successful outcomes for patients with systemic infections (5).Efforts aimed at the species-specific detection of bacterial DNA have been made (1-4, 6, 8, 9). This approach would require several PCR experiments in series or in parallel, which would be expensive and time-consuming. Therefore, the aim of this study was to introduce a prototype system for the detection of bacterial DNA that enables a hands-on time of less than 4 h, including the time for the preparation of DNA and evaluation of the PCR results. For this purpose we developed a prototype rapid real-time PCR protocol for the amplification of bacterial DNA from biological fluids. This approach enables Gram stain classification with the goal of the reliable detection and differentiation of significant pathogens in the intensive care unit (ICU) by means of fluorescence hybridization probes with calculated mismatches and melting-curve analysis in a one-run experiment. MATERIALS AND METHODS Whole organisms of 17 ICU-relevant bacteria species (Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Proteus vulgaris, Haemophilus influenzae, Enterobacter aerogenes, Enterobacter cloacae, Serratia marcescens, Bacteroides fragilis, Acinetobacter baumannii, Legionella pneumophila, Stenotrophomonas maltophilia, Enterococcus faecalis, Enterococcus faecium, Streptococcus pyogenes, Staphylococcus epidermidis, Staphylococcus aureus)were spiked in different biological fluids (water, plasma, urine), and the DNA was extracted and used for subsequent experiments. A quantitative PCR was performed on a real-time PCR instrument (LightCycler; Roche) with broad-range primers for amplification of a highly conserved region of the bacterial 16S DNA locus. The PCR products were hybridized with two fluorescence dye-labeled hybridization probes that specifically bind only to either gram-positive or gram-negative bacteria. The different fluorescence signals (640 and 705 nm, respectively) were detected by the LightCycler instrument and enabled differentiation of bacteria according to their Gram stain classificati...

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