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United States Department of Agriculture

Agricultural Research Service

Title: Use of Automated Rep-Pcr for Typing of Food-Borne Bacterial Pathogens from Avian Species

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

Submitted to: International Meeting on Microbial Epidemiological Markers
Publication Type: Abstract Only
Publication Acceptance Date: March 29, 2005
Publication Date: May 11, 2005
Citation: Line, J.E., Hiett, K.L., Siragusa, G.R., Stern, N.J., 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 avian species [abstract] International Meeting on Microbial Epidemiological Markers. p. 25.

Technical Abstract: Molecular sub-typing of pathogens has become 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) or multi-locus sequence typing (MLST) require many person-hours and are expensive. Consequently, our laboratory initiated investigations utilizing semi-automated repetitive extragenic palindromic-PCR (rep-PCR) to sub-type human food-borne pathogens that commensally colonize chickens. Prokaryotic and eukaryotic genomes contain dispersed repetitive sequences separating single copy genes. Interspersed repetitive sequences are relatively short (<500bp) and comprise a substantial portion of a genome. Primers can be synthesized complementary to repetitive motifs that allow for binding to unique species-specific sequences. Subsequent amplification utilizing rep-PCR primers could potentially provide reproducible, unique gel electrophoresis patterns. Gels can be replaced by separation of the amplification product utilizing a micro-fluid, chip-based technology amenable to an internet accessible database. Previously it was determined in our laboratory that Salmonella spp. could be classified according to serotype, while Clostridium perfringens and Campylobacter spp. could be sub-typed more precisely to individual isolate groups by gel-based rep-PCR. Specifically, rep-PCR of C. perfringens genomic DNA was more discriminatory than riboprinting. A well-established method for phylogenetic characterization of C. jejuni is comparison of flaA gene sequences encoding the short variable region (SVR). Comparison of C. jejuni phylogenetic relationships obtained by flaA SVR DNA sequences with those obtained by rep-PCR were essentially equivalent. Furthermore, the C. jejuni isolates could be geographically sub-typed into Icelandic versus U.S. clades, with the U.S. isolates separating further by regions of the country. Following rep-PCR of Campylobacter spp., genomic DNA virtual gel images were computer generated utilizing the micro-fluid, chip-based technology wherein relationships among isolates could be expressed as a dendogram or as a scatter plot. The analyses of several C. jejuni 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: 9/2/2014
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