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 (2.5×104 CFU/mL blood), the second contained 10 copies (5×103 CFU/mL blood), the third 5 copies (2.5×102 CFU/mL blood) and the fourth 2 copies (5×102 CFU/mL blood). The red blood cells were disrupted by lysis buffer [35], 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 [36]. 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 [9] and amplify the 16S Captisol nmr rRNA sequence. Nepicastat mw The PLK2 reverse primer was modified and used without the inner fluorescence labelling. Originally, the labelled primer excited the Gram JPH203 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 [10]. 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 [10] 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 [37]. Mastermixes/excitation dyes Different, non-specific intercalating dyes are used for real-time PCR investigations. Most of these are Metalloexopeptidase 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.