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United States Department of Agriculture

Agricultural Research Service

Title: Use of Automated Repetitive-Sequence-Based Pcr for Typing of Food-Borne Bacterial Pathogens with Special Emphasis on Campylobacter Spp. from Avians.

item Hiett, Kelli
item Siragusa, Gregory
item Stern, Norman
item Line, John
item Kuntz, Robin
item Seal, Bruce

Submitted to: Campylobacter Helicobacter and Related Organisms International Workshop
Publication Type: Abstract Only
Publication Acceptance Date: June 15, 2005
Publication Date: September 6, 2005
Citation: Hiett, K.L., Siragusa, G.R., Stern, N.J., Line, J.E., Kuntz, R.L., Seal, B.S. 2005. Use of automated repetitive-sequence-based pcr for typing of food-borne bacterial pathogens with special emphasis on campylobacter spp. from avians.. Campylobacter Helicobacter and Related Organisms International Workshop. p. 11.

Technical Abstract: Molecular sub-typing of pathogens is increasingly utilized to precisely characterize agents for epidemiological analyses during disease outbreaks. Most reliably utilized nucleic acid based techniques such as pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) require many person-hours and are costly. Consequently, our laboratory initiated investigations to determine if semi-automated repetitive extragenic palindromic-PCR (rep-PCR) was a viable method for sub-type analysis of Campylobacter spp. The rep-PCR subtyping technique was optimized for Campylobacter spp. and used for comparison with the established genotyping methods of flaA short variable region (SVR) DNA sequence analysis and PFGE. Fingerprints were generated from a library of Campylobacter spp. isolates recovered from a variety of epidemiologic backgrounds and sources. Analysis and phenogram tree construction using the unweighted pair group method with arithmetic mean (UPGMA) of the generated fingerprints placed isolates into epidemiologic relevant groups. Phenograms generated from rep-PCR patterns were compared with phenograms generated from flaA SVR DNA sequence analysis and PFGE analyses of the same isolates. Comparison of phylogenetic relationships obtained by flaA SVR DNA sequences with those obtained by rep-PCR were essentially equivalent. Furthermore, the Campylobacter spp. isolates could be geographically sub-typed into epidemiologically relevant clades. The analyses of several Campylobacter spp. isolates were highly reproducible following chip-based rep-PCR, and a computer accessible data base is currently being generated by our laboratory. It is our hypothesis that speciation of various unknown or Campylobacter spp. may be possible with this technology.

Last Modified: 4/20/2015
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