After vortexing, the samples were boiled in a water bath for 10 min and subsequently refrigerated at 4 °C for 10 min. Finally, the samples were centrifuged at 16 000 g for 2 min and 100 μL of the

supernatant was used as template DNA. All samples were immediately used for multiplex http://www.selleckchem.com/products/fg-4592.html and real-time PCR assays after preparation. The PCR assay was optimized using an MJ PTC 100 thermocycler (Bio-Rad, Hercules, CA). The primer sets for the PCR assay are listed in Table 2. All primers were synthesized by Integrated DNA Technologies (IDT, Coralville, IA). The reactions resulted in a 90-bp fragment for C. jejuni, a 150-bp fragment for E. coli O157:H7 (Sharma et al., 1999), and a 262-bp fragment for S. Typhimurium. (Cheng et al., 2008). The Campylobacter spp. primers were designed

by targeting a conserved region of the hsp60 gene. Reactions specific for each pathogen were first carried out independently and each reaction consisted of a 25-μL total volume mixture with 12.5 μL of SYBR Green Premix Ex Taq™ (Takara, Fisher Scientific, Pittsburg, PA), 800 nM of each primer, 1.6 μL of bovine serum albumin (BSA, 20 mg mL−1), 1 μL of DNA template, and water to volume. After BGB324 each PCR reaction was optimized independently, an m-PCR reaction was optimized to detect all three pathogens simultaneously and three independent experiments were performed to verify the reproducibility. The m-PCR reaction consisted of 25 μL of total volume mixture with 12.5 μL of SYBR Green Premix Ex Taq™ (Takara, Fisher Scientific), 400 nM of Campylobacter spp.-specific primers, 400 nM of E. coli O157:H7-specific primers, 960 nM of Salmonella spp.-specific primers, 1.6 μL of BSA (20 mg mL−1), 3 μL of three DNA templates, and water to volume. The PCR reaction was optimized to conditions of 94 °C for 2 min. and then 35 cycles

of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, with a final extension cycle at 72 °C for 5 min. The PCR products were separated in a 2% agarose gel at 100 V for 20 min. Gels were stained with ethidium bromide (10 mg mL−1) and viewed using a UV transilluminator. The SYBR green real-time PCR assay was optimized using an Eppendorf Masterplex thermocycler ep (Eppendorf, Westbury, NY). Gradient Technology in the Eppendorf unit was used to optimize annealing and extension temperatures and times. Real-time PCR assays were conducted oxyclozanide as three independent experiments and triplicate samples per experiment. The same primer sets utilized for conventional PCR, listed in Table 1, were also used for the SYBR green real-time PCR reaction. A 25-μL total volume reaction mixture consisted of 12.5 μL of SYBR Green Premix Ex Taq™ (Takara, Fisher Scientific), 800 nM of each primer, 1.6 μL of BSA (20 mg mL−1), 1 μL of DNA template, and water to volume. The PCR reaction was optimized to the conditions of 95 °C for 2 min., followed by 40 cycles of 95 °C for 15 s, 55 °C for 15 s, and 68 °C for 20 s, with fluorescence being measured during the extension phase.