Isolates were identified with a previously described mPCR assay [17; 34; 35], and a newly developed mPCR comprised of two sets of primers, one PND-1186 in vitro targeting the glyA gene of C. jejuni and the other targeting the ask gene of C. coli. Gene sequences downloaded from NCBI Selleckchem MK-8931 GenBank were aligned and analyzed using Molecular Evolutionary Genetics Analysis (MEGA) software [36] and primers were designed with the Integrated DNA Technologies PrimerQuest software. (Integrated DNA Technologies http://www.idtdna.com) The sequences of the primers are shown in Table
4. C. jejuni ATCC (American Type Culture Collection) 700819 and C. coli ATCC 43473 were used as control strains to set up the PCR conditions. The annealing temperatures of these primers were optimized with a gradient PCR program of a DNA ENgine® Thermal Cycler (Bio Rad laboratories, Hercules, CA),
and the final conditions for this mPCR assay were 20 cycles of 94°C for 30 seconds; 63°C for 1 minute and 72°C for 1 minute. Amplified products were detected by standard gel electrophoresis in 1.5% agarose (Ultra Pure DNA Grade Agarose, Bio-Rad Laboratories) in tris-borate-EDTA buffer at 100 V for 40 minutes. DNA bands in the gels were stained with ethidium bromide and visualized using a VersaDoc™ Imaging System (Bio-Rad Laboratories). Table 4 Primers developed in this study for the specific identification of C.jejuni and C. coli. Target MLN2238 Gene Primer Name Sequence (5′-3′) Tm (°C) G+C Content (%) Product Size (bp) glyA F-JK TGGCGGACATTTAACTCATGGTGC 59.6 50 264 R-JK CCTGCCACAACAAGACCTGCAATA 59.5 50 ask F-JK GGCTCCTTTAATGGCCGCAAGATT 59.8 50 306 R-JK AGACTATCGTCGCGTGATTTAGCG 58.5 50 Typing of Campylobacter isolates with PFGE Isolates from 31 samples for which
both subsamples were positive were randomly selected for PFGE analysis. Campylobacter isolates were typed using pulsed-filed gel electrophoresis (PFGE) following previously described protocols [16; 23]. Briefly, DNA was digested with SmaI and separated using a CHEF DR II system (Bio-Rad Laboratories, Hercules, CA) on 1% agarose gels (SeaKem Gold agarose; Lonza). The DNA size marker used in the gels was Salmonella enterica subsp. enterica very serovar Braenderup strain H9812 (ATCC BAA-664) restricted with XbaI. Restriction enzymes were purchased from New England BioLabs (Ipswich, MA). Gels were stained and visualized as described above (mPCR assays) and TIFF images were loaded into BioNumerics version 6 (Applied Maths, Austin, TX) for analysis. Pairwise-comparisons were done with the Dice correlation coefficient, and cluster analyses were performed with the unweighted pair group mathematical average (UPGMA) clustering algorithm. The optimization and position tolerance for band analysis were set at 2 and 4%, respectively, and similarity among PFGE restriction patters was set at 90%.
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