Direct Real-Time PCR Quantification of Campylobacter jejuni in Chicken Fecal and Cecal Samples by Integrated Cell Concentration and DNA Purification

Evaluation of the cell concentration and DNA purification assay with five different C. jejuni strains. The bacteria were spiked directly in bacterial binding and washing buffer (Genpoint), with subsequent analysis by 5′-nuclease PCR. A 10-fold dilution series, from approximately 10² to 10⁶ CFU/ml, is shown. The ΔC_T value were obtained by subtracting the C_T value for each dilution from the C_T value obtained for the log signal for approximately 6 log₁₀ CFU/ml. The following C. jejuni strains were analyzed: •, LCD 8617; ○, ATCC 43438; ×, ATCC 43436; *, ATCC 43439; and +, ATCC 43442. A linear regression curve is shown.

Effect of BSA as a PCR facilitator on cecal content samples. DNAs (2.5%) isolated from 12 mg of cecal samples were tested in 50-μl PCRs with the addition of different concentrations of BSA. Dilution series from 10³ to 10 CFU/ml were used for this analysis. The products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining and UV transillumination. mw, molecular weight marker; nt, nucleotides; neg, negative control.

PCA of the comparison of methods. The data presented in Table 2 were subjected to PCA (see Materials and Methods for details). (A) Score diagram for the methods tested. (B) Loading for the different parameters tested. The two PCs (PC1 and -2) explain 50 and 38% of the variance in the data, respectively. AMPEFF, amplification efficiency; REGRESSCO, squared regression coefficient; DETECTLIM, detection limit; time, performance time; CENTRIFUG, centrifugation; PRIMDIMER, primer dimer; BUGSB, Bugs'n Beads; DNADIR, DNA DIRECT.