DISCRIMINATION OF CAMPYLOBACTER SPP. BY USE OF GEL-BASED REP-PCR AS COMPARED WITH FLAA GENE SHORT VARIABLE REGION DNA SEQUENCE ANALYSIS

Authors: Hiett, Kelli; Siragusa, Gregory; Seal, Bruce

Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/24/2006
Publication Date: 7/24/2006
Citation: Hiett, K.L., Siragusa, G.R., Seal, B.S. 2006. Discrimination of campylobacter spp. by use of gel-based rep-pcr as compared with flaa gene short variable region dna sequence analysis. Journal of Applied Microbiology. 3:50-54.

Interpretive Summary: Campylobacter spp. are the leading causes of human bacterial food-borne illness in the United States and is commonly associated with normal poultry gastrointestinal flora. Handling and consumption of poultry or poultry related products are considered to be a primary source for this bacterial food-associated disease agent in humans. Consequently, quick and accurate, inexpensive methods are needed to trace bacterial food-borne disease agents. The unique technology of Repetitive Extragenic Palindromic-Polymerase Chain Reaction (rep-PCR) is a PCR-based method that targets known conserved, DNA sequences that are repeated in the DNA and usually present in multiple copies in bacterial genomes. This technique was used to produce easily identifiable bands that were visualized in agarose gels. The gel patterns produced from the DNA of different Campylobacter isolates correlated well with data obtained by sequencing a specific gene of the bacteria that corresponded to the bacterial flagella. Also, the time needed to obtain the gel patterns was far less than the time needed to characterize bacterial isolates utilizing other techniques such as DNA sequencing. Consequently, the unique rep-PCR technique could be used to identify sources of bacterial food-borne disease agents quickly, accurately and in a very timely fashion relative to other assays.

Technical Abstract: The rep-PCR typing technique, which targets repetitive extragenic DNA sequences in a polymerase chain reaction, was optimized for Campylobacter spp. These data were then used for comparison with the established genotyping method of flaA gene short variable region (SVR) DNA sequence analysis for molecular epidemiology. Uprime Dt, Uprime B1, or Uprime RI primers were utilized to generate gel-based fingerprints from a set of fifty Campylobacter spp. isolates recovered from a variety of epidemiologic sources. Analysis and phenogram tree construction, using the unweighted pair group method with arithmetic mean (UPGMA), 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 epidemiologic relevant groups. Genetic stability of the rep-PCR Uprime Dt patterns under non-selective, short term transfer conditions revealed a Pearson correlation approaching 99%. These same fifty Campylobacter spp. isolates were analyzed by flaA SVR DNA sequence analysis to obtain phylogenetic relationships. 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. These results indicate that rep-PCR analysis performed with the Dt primer set is a useful and effective tool for effective and accurate differentiation of Campylobacter spp. for epidemiologic analyses.