), according to the manufacturer’s instructions and quantified fluorometrically. Based on the p-Drive plasmid (3.85 kbp) plus amplicon size (variable), the concentration
of plasmid copy numbers were calculated and diluted in 1 × TE for use in quantitative real-time PCR. To ensure the standards encoded appropriate resistant gene segments, each plasmid insert was commercially sequenced (Macrogen, South Korea) and the sequence analyzed by the BLAST feature of PubMed Nucleotide data base. Absolute quantitative real-time PCR was performed to analyze total DNA extracted from fecal deposits. For real-time PCR, a Mastercycler ep Realplex (Eppendorf) was used. The conditions were: 95°C for 3 min; 40 cycles of 95°C for #selleck compound randurls[1|1|,|CHEM1|]# 30 sec, respective annealing temperatures for 30 sec, 72°C for 1 min. Each PCR (25 μL) contained (final concentrations): 1 × iQ SYBR Green Supermix (Bio-Rad Laboratories), 0.4 μM each primer, this website and 0.1 μg μl-1 BSA (New England
Biolabs, Pickering, ON). For tet (C) PCR, BSA was omitted from the reaction because of background contamination in the BSA. To each PCR, 20 ng of DNA was added. For quantification of resistant gene copy numbers, standards were prepared for each gene using the respective p-Drive plasmid containing inserted amplicons and concentrations of 106, 105, 104, 103, and 102 copies per reaction (in duplicate). Melt curve analyses were preformed on all PCR reactions to ensure specific amplification. The temperature
range was 60°C to 95°C and fluorescence was measured at 0.2°C intervals. DGGE DNA (200 ng) from replicate (n = 3) fecal deposits on days 7, 28, 56, 98, 112, and 175 were combined and used for PCR-DGGE analysis. The V6-V8 region of 16S-rRNA was amplified using primers and PCR conditions described previously . Amplified PCR-fragments were quantified fluorometrically as described above and 400 ng were loaded onto a polyacrylamide gel for electrophoresis using a D-Code system (Bio-Rad Laboratories) according to Huws et al., with the following modifications: 6% polyacrylamide with a 40-65% gradient and electrophoresis for 20 h at Org 27569 55°C, 40 V. To normalize gels for statistical analysis, a standard was made containing pooled DNA from all treated and control samples on days 7 and 175 and run every six lanes resulting in two standards per gel. Statistical Analysis Gene copy numbers were log-transformed prior to statistical analysis. The persistence of genes over time was analyzed using the Mixed procedure of SAS . Pen was considered the experimental unit. The model included the fixed effects of treatment (A44, AS700, T11, control), time (day of sampling), and the interaction between treatment and time. The repeated statement was applied to the day of sampling, using the pen nested within treatment as the subject. Various error structures were tested, and the one giving the lowest Akaike information criterion was chosen for analysis.