The plate was washed and substrate (SIGMAFAST™ p-nitrophenyl phosphate tablets
N2770, Sigma-Aldrich) was added (100 μl/well). The color was allowed to develop for 45 min in darkness and the optical density was determined using a microplate reader with a filter at 405 nm (Multiskan Ascent, Thermo Electron Corporation). Absorbance values (mean of triplicate wells) were plotted GS-4997 supplier against toxin concentrations, and values were determined from linear regression. The detection limit was at 0.31 ng/ml of SEA. Nucleotide sequence analysis The sea nucleotide sequences of six S. aureus strains (MRSA252 [GenBank: BX571856], MSSA476 [GenBank: BX571857], Mu3 [GenBank: AP009324], Mu50 [GenBank: BA000017], MW2 [GenBank: BA000033], and GSK2399872A solubility dmso Newman [GenBank: AP009351]) were retrieved from GenBank (http://www.ncbi.nlm.nih.gov/Genbank/index.html April 2009) and pairwise aligned using BioEdit v. 184.108.40.206 (Ibis Biosciences; Carlsbad, CA). DNA sequences (8 kb) upstream and downstream of the sea gene were also compared. check details The sea genes of all six strains have previously been annotated. Conventional PCR Primers were designed to confirm the results of the nucleotide sequence analysis of sea and regions adjacent to the gene
(Table 1). Two primer pairs were designed to distinguish between the two groups of nucleotide sequences, sea 1 and sea 2. Six primer pairs were designed to validate sequence differences found between strains in regions upstream and downstream of the sea gene. All primers were ordered from MWG Biotech AG. Genomic DNA from S. aureus Mu50, MW2, Newman, and SA45 was used Fludarabine chemical structure as template. The total volume of PCR mixture was 50 μl including 200 ng template DNA. The PCR mixture consisted of 1 × PCR buffer, 2 mM MgCl2, 0.2 mM each of dATP, dTTP, dCTP, and dGTP, 0.2 μM
each of forward and reverse primer and 2 U Tth DNA polymerase. All reagents except primers were obtained from Roche Diagnostics GmbH. The water used was autoclaved ultrapure water. In order to detect the amplification of possible contaminants, a negative control consisting of water instead of DNA was added to the PCR. The following PCR protocol was used: initial denaturation at 94°C for 4 min, followed by 30 cycles of denaturation at 94°C for 30 s, primer annealing at 47-55°C (see Table 1) for 30 s, and extension at 72°C for 1 min, with a final extension step at 72°C for 5 min. All amplifications were carried out using the Gene Amp 9700 thermal cycler (Perkin-Elmer Cetus; Norwalk, CT). The PCR products were visualized using 0.8% agarose (Bio-Rad Laboratories, Hercules, CA) gel electrophoresis according to Sambrook and Russell . Acknowledgements This work was supported by grants from the Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning (FORMAS) and by PathogenCombat, part of the European Commission’s 6th Framework Programme.