DNA extraction from bacterial cultures Genomic DNA from each bacterial culture was extracted using the Nucleospin® Tissue mini-kit (Macherey Nagel, Hoerdt, France) and according to the manufacturer’s instructions. The concentration of isolated double stranded DNA was determined by measuring

the optical density at 260 nm with the Spectronic® Genesys™ 5 (Spectronic Instruments Inc., New York, USA). The purity was assessed by the examination of PF-6463922 concentration 260/280 nm optical density ratios [53]. All DNA samples classified as pure (i.e. having a 260/280 nm optical density ratio between 1.8 and 2.0) were adjusted to 20 ng μL-1 in TE buffer (10 mmol Tris-HCl, 1 mmol EDTA, pH 7.6) and stored at -20°C until required for analysis. Construction of the standard curves with purified genomic DNA Total genomic DNA of C. jejuni NCTC 11168 and C. coli CIP 70.81 cultures were extracted as described above. The genome copy numbers of C. jejuni and C. coli in 100 ng of DNA (for one PCR reaction) was calculated on the basis of the genome size (1 640 Kbp for C. jejuni, 1 860 Kbp for C. coli) [54–56] and was equal to 5.2 × 107 and 4.6 × 107 copies respectively. After DNA quantitation by spectrofotometrical analysis with the Spectronic® Genesys™ 5, 10-fold dilutions of each extract were produced in TE buffer, representing 101 to 108 genome copies of C. jejuni GS-9973 clinical trial per 5 μL of https://www.selleckchem.com/products/elacridar-gf120918.html template (PCR reaction) and 0.3 × 101 to 3.0 × 108 genome

copies of C. coli per 5 μL of template (PCR reaction). Moreover, a standard curve with roughly equal genome copies of C. jejuni and C. coli together was produced for each PCR assay. Serial DNA dilutions were aliquoted: some were stocked at 4°C to be use directly, others were stored frozen at -20°C and thawed once for use. Sample collection Campylobacter-negative samples Fifteen Campylobacter-negative faecal samples were obtained from specific pathogen-free (SPF) sows and piglets from the high-security barn at the Anses centre (Ploufragan, France). Moreover, five Campylobacter-negative feed samples and 10 Campylobacter-negative environmental samples were collected from

the same high-security barns. These samples were used to test the many specificity and/or the analytical sensitivity of the real-time PCR assays. For the environmental samples, each pen of pigs was sampled on the bottom of the wall and pen partitions using swabs (sterile square pieces of cotton cloth (32 . 32 cm) moistened with isotonic saline solution) (Sodibox, La Forêt-Fouesnant, France). The swabs were placed in a sterile bag before to be analyzed. Additional faecal, feed, and environmental samples Faecal samples were obtained from both pigs experimentally inoculated with 5 × 107 CFU of Campylobacter (n = 119, respectively 67 C. coli and 52 C. jejuni faecal samples) [57] and naturally contaminated pigs in five conventional herds (n = 146).