1 M Tris-HCl-EDTA and Triton-X100 buffer at pH 8.0 (Goldenberger et al., 1995). All PCR reactions were performed with 1 μL (approximately 5–20 ng) of extracted DNA, 1 μM of each primer, 12.5 μL 2 × PCR Master

Mix (Fermentas, Le Mont-sur-Lausanne, Switzerland), and distilled DNase-free H2O (Fermentas) to a final volume of 25 μL. Oligonucleotides were obtained from Microsynth (Balgach, Switzerland). The PCR assay was performed in a Biometra® TGradient Cycler (Biolabo, Châtel-St-Denis, Switzerland) according to the following protocol: initial denaturation at 95 °C for 3 min followed by 35 cycles of 30 s denaturation at 95 °C, 30 s annealing at 62 °C, and 60 s replication at 72 °C. A final replication

was performed at 72 °C for 7 min. The reaction was subsequently cooled to 4 °C selleck inhibitor until analysis. Successful PCR products were purified using the GFX DNA purification kit (GE Healthcare Europe, Glattbrugg, Switzerland). Restriction enzymes for the RFLP assay were obtained from New England Biolabs (NEB, Ipswich, MA) and used according to specifications. Reaction volumes and purified PCR products were adjusted to a final volume of 11.5 μL per reaction and digested at 37 °C for 2 h. Enzymes were used at a final concentration of 2 and 3 U μL−1 for XbaI and MseI, respectively. Restriction digestions were performed separately for XbaI and MseI on aliquots of the original purified PCR product. Amplified DNA and RFLP Nintedanib datasheet products were analyzed by 1% and 2% agarose gel electrophoresis (Euroclone, Milan, Italy), respectively.

DNA fragments were visualized with ethidium bromide staining (2.5 mg L−1). A 100-bp TriDye DNA standard (BioConcept, Allschwil, Switzerland) was used as DNA size marker. The identification of all SBSEC reference strains (Table 1) as well as 192 S. infantarius and five S. gallolyticus isolates was successfully performed using the multiplex PCR/RFLP assay developed in this study. The specificity of the multiplex PCR assay was confirmed with various streptococcal Cobimetinib species closely related to the SBSEC as well as other LAB often present in raw milk products (Table 1). The PCR assay yielded the desired 1.1-kb fragment only with DNA of SBSEC strains corresponding to the expected product of 1119–1120 bp (Fig. 3a). It did not yield false-positive amplification of non-SBSEC reference strains or dairy isolates of closely related species commonly detected in raw milk products, such as enterococci, lactococci, and other streptococci. Especially, S. agalactiae (group B streptococci) and group C streptococci regularly detected from milk of mastitic animals (Younan & Bornstein, 2007; Whiley & Hardie, 2009; Jans, 2011) were in silico evaluated to yield a potentially false-positive result when using other assays such as the 324-fold degenerate groESL primers (Chen et al., 2008).