coli real-time PCR (R2 = 0 94) and for C jejuni real-time PCR (R

coli real-time PCR (R2 = 0.94) and for C. jejuni real-time PCR (R2 = 0.86). Among the PCR-culture positive samples for the experimentally infected pig, 72.5% of the samples had a difference in cell

number of less than 1 log, 25% of less than 2 logs, and 2.5% of less than 2.5 logs for C. coli real-time PCR assay. For C. jejuni real-time PCR assay, the results obtained by real-time PCR matched equally the results obtained by culture: 67% of the samples had a difference in cell number of less than 1 log, 29% of less than 2 logs, and 4% of less learn more than 3 logs. Figure 4 Correlation between real-time PCR and microaerobic culture for faecal samples of Campylobacter experimentally infected pigs. Scatter plot showing the differences and correlations between the real-time PCR and the microaerobic culture method for the faecal samples of pigs experimentally infected with Campylobacter for the detection of (a) C. coli and (b) C. jejuni. Data for Campylobacter-positive samples versus Campylobacter-negative samples by both methods fall close Selleck PRI-724 to the line equivalence: a- Campylobacter-positive ( n = 40) and Campylobacter-negative

( n = 25) samples respectively with a coefficient of correlation of 0.90 (R2 = 0.90). b- Campylobacter-positive ( n = 24) and Campylobacter-negative ( n = 25) samples respectively with a coefficient of correlation of 0.93 (R2 = 0.93). Analysis of field samples of naturally contaminated pigs No C. jejuni was identified among the faecal, feed, and www.selleckchem.com/mTOR.html environmental samples from the different pig herds by conventional PCR or by our C. jejuni real-time PCR assay. Conversely, all the Campylobacter tested were identified as C. coli by both methods. The specificity and the sensitivity for the C. coli real-time PCR assay with the different field samples are reported in Table 4. Table 4 Comparison of Campylobacter

coli real-time PCR and microaerobic culture in (4.1) faecal, (4.2) feed, and (4.3) environmental samples of naturally contaminated pigs       Microaerobic culture         + – Total 4.1 Campylobacter coli detection in faecal samples   + 125 1 126 MycoClean Mycoplasma Removal Kit   Real-time PCR – 3 17 20     Total 128 18 146 4.2 Campylobacter coli detection in feed samples   + 21 1 22   Real-time PCR – 2 26 28     Total 23 27 50 4.3 Campylobacter coli detection in environmental samples   + 34 2 36   Real-time PCR – 3 47 50     Total 37 49 86 4.1 Sensitivity Se = 97.7%, Specificity Sp = 94.4%, Kappa K = 0.96 4.2 Sensitivity Se = 91.3%, Specificity Sp = 96.2%, Kappa K = 0.89 4.3 Sensitivity Se = 91.9%, Specificity Sp = 95.9%, Kappa K = 0.89 For the different field samples tested, the quantification results obtained by C. coli real-time PCR matched equally the results obtained by bacterial culture: 58% of the samples had a difference in cell number of less than 1 log, 37% of less than 2 logs, and 5% of less than 3 logs.

Figure 3A shows the expected genomic loci of ech and Hyg-GAPDH-IR

Figure 3A shows the expected genomic loci of ech and Hyg-GAPDH-IR in the genome of ech +/-/Hyg parasites. PCR analysis with the genomic DNA from the drug resistant parasites and WT CL confirmed the expected gene replacement of ech1 and ech2 genes by Hyg-GAPDH-IR (Figure 3B); no products were obtained when using WT CL gDNA as the template with primer combinations f2 and D, f2 and F, C and r2, and E and r2, whereas products of the expected sizes, 1759 bp, 2178 bp, 2696 bp and 2889 bp, respectively, were observed with gDNA from ech +/-/Hyg as the template. Southern blot analysis of EcoR I digested gDNA using the ech1 gene as a probe (Figure 3A and 3C right panel) showed a 4880

bp band corresponding to the replaced allelic copy of both ech genes was undetected in ech +/-/Hyg, whereas the 3490 bp and 1365 bp bands corresponding to the second allele were retained. In addition, a 2988 bp band #CX-6258 in vivo randurls[1|1|,|CHEM1|]# and a 1478 bp band corresponding to the inserted Hyg-GAPDH-IR were observed in BanI

digested gDNA of only the ech +/-/Hyg, but not that of WT CL (Figure 3A and 3C left panel). Taken together, these results confirmed that one copy of each of the tandem ech1 and ech2 genes was replaced by the MS/GW Hyg-GAPDH-IR knockout cassette. Similarly, using linearized DNA from pDEST/ech_Neo-GAPDH (Additional file 4: Figure S3B), we generated ech +/-/Neo parasites with one copy of both ech1 and ech2 gene replaced by Neo-GAPDH-3′UTR knockout cassette (Figure 4A). This result SYN-117 mouse is confirmed by both PCR amplification

(Figure 4B) of gDNA of the drug resistant parasites, as PCR with primer combinations f2 and B, and f2 and H generated 1494 bp and 1949 bp bands respectively only PtdIns(3,4)P2 in drug resistant parasites. Southern blot hybridization also showed a 3884 bp Neo gene band in the ech +/-/Neo parasites (Figure 4C). Figure 4 Simultaneous replacement of consecutive ech1 and ech2 genes by another MS/GW construct pDEST/ ech _Neo-GAPDH. A) Diagram of ech1, ech2 and Neo-GAPDH 3′UTR genomic loci in ech +/-/Neo parasites. B) PCR genotyping analysis of: no template control (water); ech +/-/Neo (ech +/-) and WT CL (WT). See Additional file 3: Table S5 for nucleotide sequences of primers. C) Southern blot analysis of WT CL (WT) and ech +/-/Neo (ech +/-) digested with EcoRI and hybridized with Neo CDS. Diagram not to scale. Numbers are sizes (bp) of expected products. One-step-PCR knockout strategy fails to delete dhfr-ts and ech genes Since we demonstrated that at least one allele of the dhfr-ts can be deleted using the MS/GW based system, we next tested if this gene can be deleted using the one-step-PCR strategy. Transfection and selection of parasites with the knockout cassette LP-dhfr-ts-Neo failed to yield drug resistant parasites, despite 4 independent attempts.

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