Ultraclean bloodspin kit

Pre-enriched blood samples were centrifugated in a macrocentrifuge at 3000 x g and the supernatant pipetted out leaving approximately 200 μl to resuspend the pellets. DNA from blood, including bacterial genomic DNA, was extracted following the UltraClean BloodSpin Kit (MO BIO Laboratories CA USA) extraction protocol. DNA was eluted in a final volume of 100 μl and stored at -20°C. A 40-cycle real-time PCR was performed using a mastermix of Platinum Quantitative UDG reagents on an ABI 7500. Three primer pairs (Table 1) designed using Primer Quest, were added in monoplex reactions (S1 Table) to detect the presence of either S. Typhi or S. Typhimurium. A pan-Salmonella primer pair targeting invA was designed using sequences from S. Typhimurium ST313 D23580 (Accession FN424405) and S. Typhi CT18 (Accession AL513382). A primer pair specifically identifying S. Typhimurium was designed against the flagellin gene fliC and S. Typhi was detected using a primer pair targeting the fimbrial gene staG.

DNA was isolated from the culture using UltraClean™ BloodSpin™ Kit (MO BIO Laboratories, CA, USA) according to the manufacturer's instruction, except that the DNA was eluted with 50 μl buffer 5 preheated at 65°C and the filter unit was incubated at 65°C for 5 min before centrifugation. The aliquots (10 μl) of DNA preparation were used for PCR amplification.

DNA was extracted from blood and tissue samples either with the standard phenol–chloroform method [50 ], or using the UltraClean® BloodSpin™ Kit or UltraClean® Tissue & Cells DNA Isolation Kit (MoBio Laboratories), and from feathers using the method described in Rönkä et al. [34 (link)]. Individuals were genotyped for 12 microsatellite loci, which were amplified in 10 µl volumes containing 20–100 ng of template DNA, 0.1 µM of each primer, 0.8–1 mM MgCl₂, 0.2 mM of dNTPs, 1 µl of 10 × PCR-Buffer and 0.l U of DNA-polymerase (Biotools). The amplification profile was 94 °C for 1 min followed by 35 cycles of 94 °C for 30 s, 52–58 °C for 45 s (see Additional file 1: Table S1), 72 °C for 45 s and 72 °C for 10 min for final extension. The PCR reactions were run with ABI 3730 sequencer using GS500-Liz size standard (Applied Biosystems) and the loci were scored with GeneMapper v. 4.0. (Applied Biosystems), except for the Swedish samples, which were scored with CEQ^TM8000 Genetic Analysis System (Beckman Coulter) using the Fragment Analysis Module v. 8.0.52. Due to possible differences between the allele sizes defined by the two sequencers, samples were calibrated by genotyping five Swedish individuals with both sequencers. Genotyping error rate was calculated by amplifying most individuals twice. If differences were found between the two runs, samples were genotyped twice more.