Reference strains of 17 clinically relevant bacterial species were collected, as typical of the main causative agents of bloodstream infections . Nine reference strains, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Enterococcus faecalis ATCC29212, Listeria monocytogenes ATCC 4701, Bacteroides fragilis ATCC 25285, Pseudomonas aeruginosa ATCC 27853, Haemophilus influenzae ATCC 49247, Escherichia coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603 were from the American Type Culture Collection. [ATCC]. Streptococcus pyogenes OKI 80002 was from the National Centre for Epidemiology, Hungary [OKI] and Proteus vulgaris HNCMB 60076 was from the Hungarian National Collection of Medical Bacteria [HNCMB]. Furthermore, to confirm the reliability and reproducibility of the technique, clinical strains of S. aureus (n = 4), S. epidermidis (n = 6), S. pyogenes (n = 2), E. faecalis (n = 2), E. faecium (n = 3), L. monocytogenes (n = 1), B. fragilis (n = 2), P. aeruginosa (n = 1), H. influenzae (n = 1), E. coli (n = 5), K. pneumoniae (n = 5), P. vulgaris (n = 3), Stenotrophomonas maltophilia (n = 2), Serratia marcescens (n = 2), Enterobacter aerogenes (n = 2), E. cloacae (n = 2) and Acinetobacter baumannii (n = 3) from the Institute of Clinical Microbiology at the University of Szeged were also included. The species identities of the clinical isolates were confirmed by conventional biochemical methods.
Ten fungal strains were examined. Five reference strains, Candida albicans ATCC 10231 and ATCC 14053, C. tropicalis ATCC 750, C. parapsilosis ATCC 22019 and C. glabrata ATCC 39316, were from the [ATCC], Cryptococcus neoformans IFM 5844 and IFM 5855 were from IFM Quality Services Pty Ltd [IFM], and Aspergillus fumigatus SzMC 2486, A. flavus SzMC 2536 and A. niger SzMC 2761 were from the Szeged Microbiological Collection [SzMC]. Furthermore, clinical strains of C. albicans (n = 14), C. glabrata (n = 5), C. tropicalis (n = 4), C. parapsilosis (n = 5), C. krusei (n = 4), C. quillermondii (n = 4), C. lusitaniae (n = 3), C. norvegensis (n = 1), C. inconspicua (n = 2), C. dubliniensis (n = 2) and Cryptococcus neoformans (n = 2) from the Institute of Clinical Microbiology at the University of Szeged were also tested.
Bacterial DNA purification
The bacterial strains were grown on Columbia agar base under aerobic conditions, except that Bacteroides fragilis was grown under anaerobic conditions. The bacterial DNA was extracted with the QIAamp® DNA Blood Mini Kit (QuiaGene Inc, Chatsworth, Calif., USA), following the manufacturer’s instructions in “Protocols for Bacteria”. One millilitre of log-phase culture suspension, at a concentration of 107 CFU/mL, was used for the preparation. For determination of the sensitivity of the reaction, 100 μL of the serially diluted S. aureus reference strain was used for DNA extraction. The number of bacterial cells was determined by plating aliquots of serially diluted samples onto Columbia agar base.
For lysis of the rigid multilayered G + bacterial cell wall, we used a pre-incubation step with 20 mg/mL lysozyme (in 20 mM Tris · HCl, pH 8.0, 2 mM EDTA, 1.2% TritonX100). The spin protocol for “DNA Purification from Tissues” was followed, after incubation at 30°C for 30 min. The final concentration of DNA was 2.0-13.8 ng/μL, with a ratio A260/A280 = 1.6-1.8 after purification.
Fungal DNA purification
All the fungi were grown on Sabouraud medium. The fungal DNA was extracted from 1 mL of a log-phase culture suspension containing 9.6 × 107 of fungal cells. For determination of the sensitivity of the reaction, 100 μL of the serially diluted C. albicans reference strain was used for DNA extraction. The number of fungal cells was determined by plating aliquots of serially diluted samples onto Sabouraud-glucose medium.
We followed the QIAamp® DNA Mini Kit Protocol for Yeasts. In this case, additional reagents were required for elimination of the complex fungal cell-wall structure: sorbitol buffer (1 M sorbitol, 100 mM EDTA, 14 mM β-mercaptoethanol)  was used, and the samples were incubated with lyticase for 30 min at 30°C. Efficient and complete lysis was achieved in 1.5 hour in a shaking water-bath. This purification yielded 2.0–25 μg of DNA in 100 μL of water (2.0–13.8 ng/μL), with A260/A280 = 1.6–1.8.
DNA preparation from infected blood
Samples of 180 μL healthy donor bloods in EDTA vacutainer tubes were infected with 20 μL of log-phase culture suspension at a concentration of 108 CFU/mL bacterial and/or fungal suspensions. Bacterial and fungal cells were quantified, in a Bürker chamber, by viable counts. For the sensitivity testing of the prototype system, the bloods were infected with five dilutions of the log-phase culture suspension at a final volume of 20 μL. The first dilution contained 50 copies in 1 μL template DNA (2.5x104 CFU/mL blood), the second contained 10 copies (5x103 CFU/mL blood), the third 5 copies (2.5x102 CFU/mL blood) and the fourth 2 copies (5x102 CFU/mL blood). The red blood cells were disrupted by lysis buffer , the bacterial and fungal cell wall lysed using the freezing-thawing method. After digestion with Proteinase K, the DNA was extraction carried out as reported previously .
Bacterial and fungal primer design, FRET probes
Two primer pairs were used for multiplex amplification of bacterial and fungal DNA.
The bacterial primer pair was PLK1 (TAC GGG AGG CAG CAG) forward and PLK2 (TAT TAC CGC GGC TGC T) reverse, which are highly conserved in different groups of bacteria  and amplify the 16S rRNA sequence. The PLK2 reverse primer was modified and used without the inner fluorescence labelling. Originally, the labelled primer excited the Gram specific probes. We applied the non-specific SYBR Green dye for excitation; it also serves for visualization of the fungal amplicons. This primer-pair produces a 187 bp fragment in each species.
Previously hybridization probes were used for the Gram classification . ISN2 (5′-CCG CAG AAT AAG CAC CGG CTA ACT CCG T-3′) labelled with LCRed 640 was specific for G-, and ISP3 (5′-CCT AAC CAG AAA GCC ACG GCT AAC TAC GTG-3′) labelled with Cy5.5 was specific for G + bacteria, and the  ISP2 probe was labelled with LCRed705 at the 5′ end. The producers offered Cy5.5 dye instead of LCred705. This modified probe was used in our experiments.
The ITS86 forward (GTG AAT CAT CGA ATC TTT GAA C) and the ITS 4 reverse (TCC TCC GCT TAT TGA TAG C) primers were used for detection of the fungi. These primers amplify a 192–494 bp sequence of ITS2 region, which is a highly variable part between the 5.8S and 28S rRNA sequence .
Different, non-specific intercalating dyes are used for real-time PCR investigations. Most of these are accessible in ready-to-use, mastermix formulae. Our goal was to choose the best dye for excitation of the labelled probes. The tested dyes were LCGreen “LightCycler® 480 High Resolution Melting Master” (Roche Diagnostic GmbH, Mannheim, Germany); SybrGreen “LightCycler® 480 DNA Master SYBR Green I”, (Roche); “IQ™ SYBR® Green Supermix” (Bio-Rad Laboratries, Inc., Hercules, CA, USA); “Maxima™ SYBR Green qPCR Master Mix no ROX” (Fermentas, Vilnius, Lithuania); and EvaGreen (“LC-FastStart DNA Master Hybridization Probes” (Roche) combined with EvaGreen dye (Biotium Inc., Hayward, CA, USA) and “Sso Fast™ EvaGreen® Supermix” (BioRad). All mastermixes were used according to the manufacturer’s instructions.
PCR was performed using a LightCycler real-time PCR instrument (Roche). The reaction volume of 10 μL contained 1 μL of DNA (with a final concentration of ~10 ng/μL), 1 μM of each of the primers, 0.7 μM of each of the probes, an appropriate amount of mastermix, and 0.2 mM BSA (in the cases of the Fermentas and BioRad mastermixes).
The PCR conditions were as follows: initial denaturation at 95°C for 600 s, followed by 40 cycles of denaturation (95°C for 0 s, 20°C/s), annealing (55°C for 15 s, 20°C/s), and extension (72°C for 20 s, 2°C/s). The emitted fluorescence was measured after the annealing steps. The melting-curve analysis procedure consisted of 1 cycle at 95°C for 10 s, 40°C for 120 s, followed by an increase in the temperature to 95°C at 0.2°C/s. The fluorescence signal (F) was monitored continuously during the temperature ramp, and plotted against temperature (T).
The melting peaks were evaluated using the LightCycler Software V 3.5. The melting-peaks were determined through the manual Tm option on the three detection channels (F1, F2 and F3).
The standard deviation (SD) of the melting-points was calculated from five parallel experiments. The fungal or bacterial samples were verified by gel electrophoresis on 1.5% agarose gel, with the help of a low-range DNA ladder.
The sensitivity of the multiplex PCR calculated from five dilutions of the bacterial suspension.