0 (Applied Biosystems, Foster city, CA, USA) according to the recommendations of the manufacturer (Table 5). This software was used to choose the best combinations of each primers-probe set Selleck BMS345541 values. Finally, the selected primers and probes were checked for homology to non-target sequences by a search with the BLAST program of the National Center for Biotechnology Information (NCBI). Primers and MgB probes were synthesized by Applied Biosystems and stored at -20°C prior to use. Real-time PCR amplification Reactions were done in 20 μL PCR mixtures containing 10 μL of 1X Taqman Universal PCR
Mastermix (AmpliTaq Gold™ DNA polymerase, dNTPs, Passive reference (ROX), and optimised buffer components including 5 mM MgCl2), 400 nM of each primer (glyA-R SU5402 research buy and glyA-F for C. coli real-time PCR assay, hipO-R and hipO-F for C. click here jejuni real-time PCR assay), 200 nM of the probe (glyA-P
and hipO-P respectively), and 5 μL of template DNA. The thermal cycle protocol used was the following: activation of the Taq DNA polymerase at 95°C for 10 min, then 45 or 48 cycles of 15 s at 95°C and 60 s at 60°C. Thermal cycling, fluorescent data collection, and data analysis were carried out with the ABI PRISM® 7300 Sequence Detection System (Applied Biosystems) according to the manufacturer’s instructions. Fluorescence of FAM and VIC was measured at their respective wavelengths during the annealing/elongation step of each cycle. After real-time data acquisition, the baseline cycles for the FAM and VIC signals were set from cycle three to three cycles below the cycle at which the first signal
appeared and the threshold value at the point at which the fluorescence exceeded 10 times the standard deviation of the mean baseline emission. The threshold cycle (Ct) is the first PCR cycle at which a statistically Farnesyltransferase significant increase in fluorescent signal is detected. All reactions were carried out alongside a non template control containing all reagents except DNA, positive controls containing DNA from reference strains (C. jejuni NCTC 11168 and/or C. coli CIP 70.81), and negative controls containing DNA from Listeria monocytogenes ATCC 19115 and from Escherichia coli CIP V517. All the DNA extractions were done as described before. Each control was run in triplicate and each sample in duplicate. Evaluation of performance of the real-time PCR assays Specificity and sensitivity The specificity of each real-time PCR assay was first assessed with purified genomic DNA preparations (about 106 genome copies per PCR reaction) of different bacterial strains (Table 1) and then with DNA extracted from 30 Campylobacter-negative faecal, feed, and environmental samples as defined above. This screening strategy, described previously by Lagier et al. (2004) , ensure the specificity of the primers and probes for C. jejuni and C. coli only in field samples.