Figure 2 Dynamic range and sensitivity of the Campylobacter coli and Campylobacter jejuni real-time
PCR assays with samples containing roughly equal GW786034 price genome copies of both species. The linear range of each real-time PCR assay was determined using C. coli CIP 70.81 and C. jejuni NCTC 11168 standard DNA together. Standard curves of 10-fold serial dilution of both C. coli and C. jejuni standard DNA (roughly from 101 to 108 genome copies of each species per PCR reaction) by (a) C. coli real-time PCR assay and by (b) C. jejuni real-time PCR assay are reported, each dot representing the result of duplicate amplification of each dilution. The coefficients of determination and the slopes of each regression curve are indicated. The standard curves are obtained by correlation of the threshold cycle values (Ct) and log10 input genome copy number (Log CO) from the amplification plot. Precision of the C. jejuni and C. coli real-time PCR assays To obtain values for the intra- and inter-assay variation of each real-time PCR assay, purified genomic DNA from 101 to 108 genome copies per PCR reaction was subjected to each real-time PCR in ten duplicates, with 10 different mixes performed on different runs. The results are presented in Table 2. The coefficients of variation (CV) of the Ct values for the ten different intra-assay find more experiments ranged from 0.81 to 2.27% for C. coli real-time PCR
and from 0.35 to 5.63% for NCT-501 C.
jejuni real-time PCR. The mean standard curves were y = -3.33x + 40.17 with R2 = 0.99 for C. coli PCR and y = -3.33x + 40.53 with R2 = 0.99 for C. jejuni PCR. The CV of the Ct values for the inter-assay variation ranged from 1.52 to 4.89% and from 0.67 to 2.65%, respectively for C. jejuni real-time PCR assays for the standard curves generated with purified genomic DNA of C. coli CIP 70.81 and C. jejuni NCTC 11168, ranging from 101 to 108 genome copies per PCR reaction (genome copy number) and with DNA extracted from Campylobacter-negative PD184352 (CI-1040) pig faecal samples spiked with different amounts of C. coli and C. jejuni ranging from 2 × 102 to 2 × 107 CFU/g of faeces including the DNA extraction procedure (CFU/g of faeces) Intra-assay 1 Inter-assay 2 C. coli C. jejuni C. coli C. jejuni Genome copy number CV c (%) Ct range CV j (%) Ct range CV c (%) Ct range CV j (%) Ct range 10 8 2.27 14.18-15.25 5.63 14.18-17.15 4.89 13.86-16.11 1.94 14.30-15.01 10 7 1.33 16.63-17.71 0.95 17.55-18.21 4.69 16.33-17.88 0.83 17.86-18.27 10 6 1.99 20.05-20.78 1.13 21.02-21.81 3.42 19.29-21.80 1.37 21.15-22.04 10 5 1.60 23.32-24.63 0.57 24.15-24.69 4.08 23.22-25.55 0.67 24.01-24.48 10 4 0.81 26.