The PCR program consisted of an initial activation step for 15 min at 95 °C followed by 40 cycles of denaturation for 60 s at 95 °C and annealing/extension for 60 s at the optimized temperature of 59 °C. Owing to the careful selection of the two detection channels employed (FAM/TexasRed),

a colour compensation experiment was not necessary. To determine the detection limit of the duplex Selleck Venetoclax real-time PCR and the corresponding singleplex PCR, a DNA dilution series ranging from 50 ng μL−1 to 0.5 fg μL−1 was measured. Each measurement was repeated three times with DNA of E. cloacae ssp. cloacae DSM 30054T. The same dilution series was used for calculating PCR linearity and efficiencies from the formula E = 10−1/slope (Pfaffl, 2001). All isolates were grown for 20 h on Columbia sheep blood agar plates at 37 °C. Single colonies were picked and resuspended in 300 μl of sterile water. Nine hundred microlitres of ethanol abs. was added. The mixture was Pexidartinib in vitro centrifuged at 10 000 g for 2 min. After the supernatant was discarded, the pellet was centrifuged again. Residual ethanol

was completely removed by pipetting, and the pellet was allowed to dry at room temperature. Subsequently 30 μL of formic acid (70%) was added and mixed with the pellet by vortexting. Next, 30 μL of acetonitrile was added and mixed thoroughly. The solution was centrifuged at maximum speed for 2 min again and 1.5 μL of the supernatant was spotted on the MALDI target plate (Bruker Daltonics, Bremen, Germany) in two replicates. Immediately after drying, 1.5 μL of the Matrix solution was added Resminostat to each spot and allowed to air dry. The matrix used was a saturated solution of α-cyano-4-hydroxycinnamic acid (Bruker Daltonics) dissolved in 50% acetonitrile (v/v), with 0.025% trifluoroacetic acid (v/v). Brukers Bacterial Test Standard (Bruker Daltonik GmbH, Bremen, Germany) was used as mass calibration standard. Samples were then processed in the MALDI-TOF MS spectrometer (Microflex LT; Bruker Daltonics) with flex control software (Bruker Daltonics). Each spectrum

was obtained by averaging 500 laser shots acquired in the automatic mode at the minimum laser power necessary for ionization of the samples. The spectra have been analysed in an m/z range of 2–20 kDa. Data analysis was performed using BioTyper™ 1.1 software (Bruker Daltonics). MALDI-TOF identifications were classified using score values proposed by the manufacturer: a score ≥ 2 indicated species identification; a score between 1.7 and 1.9 indicated genus identification; and a score < 1.7 indicated no reliable identification. According to Mellmann et al. (2009), a score value distance of at least 0.15 between the two best-scored species was defined as necessary for a precise species identification.