Campylobacter spp. subtype analysis using gel-based repetitive extragenic palindromic-PCR discriminates in parallel fashion to flaA short variable region DNA sequence analysis.
OBJECTIVE
The repetitive extragenic palindromic-PCR (rep-PCR) subtyping technique, which targets repetitive extragenic DNA sequences in a PCR, was optimized for Campylobacter spp. These data were then used for comparison with the established genotyping method of flaA short variable region (SVR) DNA sequence analysis as a tool for molecular epidemiology.


RESULTS
Uprime Dt, Uprime B1 or Uprime RI primers were utilized to generate gel-based fingerprints from a set of 50 Campylobacter spp. isolates recovered from a variety of epidemiological backgrounds and sources. Analysis and phenogram tree construction, using the unweighted pair group method with arithmetic mean, of the generated fingerprints demonstrated that the Uprime Dt primers were effective in providing reproducible patterns (100% typability, 99% reproducibility) and at placing isolates into epidemiological relevant groups. Genetic stability of the rep-PCR Uprime Dt patterns under nonselective, short-term transfer conditions revealed a Pearson's correlation approaching 99%. These same 50 Campylobacter spp. isolates were analysed by flaA SVR DNA sequence analysis to obtain phylogenetic relationships.


CONCLUSIONS
The Uprime Dt primer-generated rep-PCR phenogram was compared with a phenogram generated from flaA SVR DNA sequence analysis of the same isolates. Comparison of the two sets of resulting genomic relationships revealed that both methods segregated isolates into similar groups.


CONCLUSIONS
These results indicate that rep-PCR analysis performed using the Mo Bio Ultra Clean Microbial Genomic DNA Isolation Kit for DNA isolation and the Uprime DT primer set for amplification is a useful and effective tool for accurate differentiation of Campylobacter spp. for subtyping and epidemiological analyses.
Hiett
K L
KL
U.S. Department of Agriculture, Agricultural Research Service, Poultry Microbiological Safety Research Unit, Russell Research Center, Athens, GA 30604-5677, USA. khiett@saa.ars.usda.gov
Seal
B S
BS
Siragusa
G R
GR
D015373:Bacterial Typing Techniques; D016123:Campylobacter jejuni / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis*; D016521:Electrophoresis, Gel, Pulsed-Field; D005408:Flagellin / Q000235:genetics; D005838:Genotype; D017720:Molecular Epidemiology / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D005408:Flagellin; C067138:flaA protein, bacteria