jejuni real-time PCR assays), each Lapatinib dilution point was tested in duplicate and the mean standard curves were used for quantity estimation. The CV of the Ct values were calculated for the ten different inter-assay experiments. They illustrate the variability of the Ct values obtained between experiments including the specific DNA extraction procedure and the amplification step. Use of the standard curves The standard curves were thus used (i) to evaluate the sensitivity of the real-time PCR assays, (ii) to assess the intra- and inter-assay variabilities, and (iii) to allow a reliable
quantification of C. jejuni and C. coli in pure cultures or in the field samples. Statistical analysis PCR amplification efficiency (E) was estimated using the slope of the standard curve and the formula E = 10(-1/slope)-1. A reaction with 100% efficiency will generate a slope of -3.32. Data analysis Gefitinib price was performed using the SDS software (Applied Biosystems).
The 119 field samples from the experimental infection were evaluated in parallel with the real-time PCR assays and the bacterial culture described in this study. All data analyses were performed with Microsoft excel and SAS Systems version 8 (SAS, Cary, N.C.). Specificity and sensitivity were assessed using the bacterial culture as a gold standard. The sensitivity was calculated as a/(a+c), where a is the number of samples found positive by both real-time PCR and bacterial culture (direct inoculation or after selective enrichment) and c is the number of samples positive by bacterial culture but negative by real-time Urease PCR. The specificity was calculated as d/(b+d), where d is the number of samples negative by both methods and b is the number
of samples positive by real-time PCR but negative by bacterial culture. Kappa-statistic was used to measure the agreement between the microaerobic cultivation and each species-specific real-time PCR assay . Acknowledgements The authors thank Sebastien Tessier for technical assistance during his practice training period and the staff of the BioEpAR and MAE units at the Veterinary School of Nantes, notably Jean-Yves Audiart, Françoise Armand, Emmanuelle Blandin, and Françoise Leray. We thank especially Francis Mégraud and Philippe Lehours of the French National Reference Center for Campylobacter and Helicobacter (Bordeaux, France) for providing us reference strains from their collection and field strains from clinical cases. This work was supported by grants from INRA, Anses, and the Region Pays de La Loire. References 1. Moore JE, Corcoran D, Dooley JS, Fanning S, Lucey B, Matsuda M, McDowell DA, Megraud F, Millar BC, O’Mahony R, O’Riordan L, O’Rourke M, Rao JR, Rooney PJ, Sails A, Whyte P: Campylobacter. Vet Res 2005,36(3):351–382.PubMedCrossRef 2. EFSA: The Community Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents, Antimicrobial Resistance and Foodborne Outbreaks in the European Union in 2006. The EFSA Journal 2007, 130. 3.