One Campylobacter isolate per positive batch was tested. For the PCR the consensus pair of primers for the flaA gene described by Wassenaar and Newell (Wassenaar and Newell, 2000 (link)) and the reagents and conditions described by Nachamkin et al. (Nachamkin et al., 1993 (link)) were applied. For restriction fragment length polymorphism (RFLP) analysis flaA PCR amplicons were treated with restriction enzyme DdeI (Thermo Scientific, West Palm Beach, FL). PCR amplicons (7 μL) were digested according to the manufacturer's instructions and then separated by electrophoresis for 1:40 hours at 120 V. The gels were stained and photographed. The relatedness among the flaA-RFLP profiles was analyzed with GelCompar II software v. 6.6 (Applied Maths, Sint-Martems-Latem, Belgium). Bands representing fragments between 200 bp and 1,100 bp in size were included in the analysis. A similarity dendrogram was constructed by the unweighted pair group method using arithmetic averages algorithm (UPGMA). DICE similarity coefficient with a tolerance position of 1% was calculated. A flaA-RFLP genotype was assigned on the basis of the difference in the presence of at least one band in the Ddel fingerprint.
Gelcompar 2 software 5 6
Manufactured by bioMérieux
Sourced in Belgium
GelCompar II software v. 6.6 is a data analysis software for gel-based microbial identification and typing. The software allows for the analysis and comparison of electrophoretic patterns, such as those obtained from techniques like pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP).
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Lab products found in correlation
2 protocols using gelcompar 2 software 5 6
1
Campylobacter flaA Gene Profiling by RFLP
2
Salmonella Serotyping and Genotyping
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One Salmonella isolate per positive sample was further characterized. To limit the number of Salmonella strains to be serotyped, isolates were grouped by an enterobacterial repetitive intergenic consensus (ERIC) PCR as described by Rasschaert et al. [18 (link)]. ERIC PCR was performed on 59 strains within the same run. Based on ERIC PCR profiles 16 isolates were selected for serotyping. All these selected isolates and the 3 isolates not included in the ERIC PCR run were serotyped according to the Kauffmann-White scheme.
To characterize the Salmonella strains within each serotype, all isolates were genotyped by pulse field gel electrophoresis (PFGE) after digestion with XbaI enzyme [19 ]. The relatedness among the PFGE profiles was analyzed with GelCompar II software v. 6.6 (Applied Maths, Sint-Martems-Latem, Belgium). Bands representing fragments between 35 kb and 1140 kb in size were included in the analysis. A similarity dendrogram was constructed by the unweighted pair group method using arithmetic averages algorithm (UPGMA). DICE similarity coefficient with a position tolerance of 1.4 was calculated. A PFGE genotype was assigned on the basis of the difference in the presence of at least one band in the XbaI fingerprint [20 (link)]. Genotypes were identified by numerical suffixes after a capital indicating the serotype (e.g. I-1 refers to serotype Infantis).
To characterize the Salmonella strains within each serotype, all isolates were genotyped by pulse field gel electrophoresis (PFGE) after digestion with XbaI enzyme [19 ]. The relatedness among the PFGE profiles was analyzed with GelCompar II software v. 6.6 (Applied Maths, Sint-Martems-Latem, Belgium). Bands representing fragments between 35 kb and 1140 kb in size were included in the analysis. A similarity dendrogram was constructed by the unweighted pair group method using arithmetic averages algorithm (UPGMA). DICE similarity coefficient with a position tolerance of 1.4 was calculated. A PFGE genotype was assigned on the basis of the difference in the presence of at least one band in the XbaI fingerprint [20 (link)]. Genotypes were identified by numerical suffixes after a capital indicating the serotype (e.g. I-1 refers to serotype Infantis).
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