2013 . DNA sequencing Purified DNA fragments were subjected to selleckchem cycle sequencing with BigDye™ Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Darmstadt, Germany). Amplification primers were also used as sequencing primers. Nucleotide sequences were determined on an ABI Prism 310 Genetic Analyzer (Applied Biosystems). Analysis of sequence data VNTR sequence data were aligned using BioEdit (Biological sequence alignment editor,
Ibis Therapeutics, Carlsbad, CA, USA). Stability testing The stability of the markers Ft-M3, Ft-M6, Ftind33, Ftind38, and Ftind49 was assessed for two F. tularensis subsp. holarctica strains that were isolated from a hare (06T0001) and a red fox (Vulpes vulpes) (10T0191), respectively. The isolates were passaged twenty times on MA-104 cells in 12.5 ml cell culture flasks (Becton Dickinson GmbH, Heidelberg, Germany). Confluent monolayers of MA-104 cells were washed with phosphate- buffered saline, pH 7.4. The bacterial suspensions or cell culture samples were inoculated on the cells at 37°C for 1 h. The inoculum was replaced with Dulbecco’s Hydroxylase inhibitor Modified Eagle’s Medium (DMEM) and incubated at 37°C in a humidified air atmosphere with 5% CO2. After incubation for 3 to 5 days when the cells PSI-7977 detached from the surface, the bacteria were harvested by two freeze-thaw cycles. The bacteria/cell suspensions were used for preparation Rolziracetam of DNA. MALDI-TOF
typing Samples were taken from single colonies, ethanol-precipitated and extracted with 70% formic acid as described by Sauer et al. . The extract was diluted with one volume acetonitrile and 1.5 μL of the mixture was spotted to a steel MALDI target. The dried extract was overlaid with 1.5 μL of a saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile/2.5% trifluoroacetic acid as matrix and was again allowed to dry. A custom-made database of reference spectra, or main spectra (MSP), was constructed
using the BioTyper software (version 1.1, Bruker Daltonics, Bremen, Germany) following the guidelines of the manufacturer. Each sample was spotted six-fold and four single spectra with 500 laser pulses each were acquired from each spot with an Ultraflex I instrument (Bruker Daltonics) in the linear positive mode in the range of 2,000 to 15,000 Da. Acceleration voltage was 25 kV and the instrument was calibrated in the range of 4,364 to 10,299 Da with reference masses of an extract of an Escherichia coli DH5-α strain prepared according to Sauer et al. . MSP were generated within the mass range of 2,500 to 15,000 Da with the following default parameters: compression of the spectrum data by a factor of 10, baseline smoothing by the Savitsky-Golay algorithm (25 Da frame size), baseline correction by 2 runs of the multi-polygon algorithm, and peak search by spectra differentiation.