Additionally, numerous non-Salmonella strains (n = 36) were shown in Table 3

for exclusivity testing, including E. coli O157:H7, non-O157 Shiga toxin-producing E. coli (STEC) strains, Shigella and other foodborne pathogen strains. Bacterial growth All bacteria were grown in Luria Bertani (LB) broth (Becton Dickinson and Company, Sparks, MD) at 37°C with shaking at 180 rpm, or as otherwise stated. Growth of Salmonella Enteritidis (SARB16) was monitored by determining the turbidity at 600 nm (OD600) using a DU530 spectrophotometer (Beckman, CA). To enumerate bacterial cells, cultures were diluted serially in 10-fold increments with LB medium and plated onto LB agar plates at 37°C overnight. DNA extraction DNA was extracted GDC-0068 cell line from bacterial cultures using the Puregene cell and tissue kit (Gentra, Minneapolis, MN) according to the manufacturer’s instructions. Briefly, signaling pathway 1 ml of overnight grown culture was centrifuged, resuspended with 3 ml of cell lysate solution, and incubated at 80°C for 5 min. Fifteen microliters of RNase A solution was added, mixed, and incubated at 37°C for 60 min. One milliliter of protein precipitation solution

was added, vortexed and centrifuged. The supernatant was combined with 3 ml of 2-propanol, mixed, and centrifuged. The pellets were washed with 70% ethanol, rehydrated with 500 l of DNA hydration solution, and incubated at 65°C for 1 h. The DNA concentrations were determined by measuring

optical density (OD260) using a spectrophotometer (NanoDrop Technology, Oxymatrine Wilmington, DE). Primers and probes The sequence of the invA gene used in this study was identified from the genomic sequence of GenBank accession number M90846. Primers and probe were designed using Primer Express© 3.0 software from Applied Biosystems Inc. (ABI, Foster City, CA). Five primer pairs that encode different lengths of amplicons were designed and are listed in Table 1. qPCR assay conditions Reaction mixtures consisted of 12.5 μl of 2 × Universal Master Mix (ABI), 200 nM of forward and reverse primers targeting invA gene in Salmonella and 100 nM of probe. Template DNA (5 μl of 20 pg/μl) and an appropriate volume of nuclease-free water (Qiagen Sciences, MD) were added to reach a final reaction volume of 25 μl. qPCR conditions were set as follows: activation of TaqMan at 95°C for 10 min; followed by 40 cycles of denaturation at 95°C for 10 s and annealing/extension at 60°C for 1 min. qPCR with internal amplification control To ensure the amplification was free of inhibitory factors from examined samples, an internal amplification control (IAC) was set. The primers and probe for IAC were designed [21, 44] based on the pUC19 DNA (Promega, Madison, MI), which was diluted to 50 fg/μl.