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Fingerprinting of Campylobacter jejuni by using resolution-optimized binary gene targets derived from comparative genome hybridization studies.

ABSTRACT: The aim of this investigation was to exploit the vast comparative data generated by comparative genome hybridization (CGH) studies of Campylobacter jejuni in developing a genotyping method. We examined genes in C. jejuni that exhibit binary status (present or absent between strains) within known plasticity regions, in order to identify a minimal subset of gene targets that provide high-resolution genetic fingerprints. Using CGH data from three studies as input, binary gene sets were identified with "Minimum SNPs" software. "Minimum SNPs" selects for the minimum number of targets required to obtain a predefined resolution, based on Simpson's index of diversity (D). After implementation of stringent criteria for gene presence/absence, eight binary genes were found that provided 100% resolution (D=1) of 20 C. jejuni strains. A real-time PCR assay was developed and tested on 181 C. jejuni and Campylobacter coli isolates, a subset of which have previously been characterized by multilocus sequence typing, flaA short variable region sequencing, and pulsed-field gel electrophoresis. In addition to the binary gene real-time PCR assay, we refined the seven-member single nucleotide polymorphism (SNP) real-time PCR assay previously described for C. jejuni and C. coli. By normalizing the SNP assay with the respective C. jejuni and C. coli ubiquitous genes, mapA and ceuE, the polymorphisms at each SNP could be determined without separate reactions for every polymorphism. We have developed and refined a rapid, highly discriminatory genotyping method for C. jejuni and C. coli that uses generic technology and is amenable to high-throughput analyses.

SUBMITTER: Price EP

PROVIDER: S-EPMC1694235 | biostudies-literature | 2006 Dec

REPOSITORIES: biostudies-literature

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Fingerprinting of Campylobacter jejuni by using resolution-optimized binary gene targets derived from comparative genome hybridization studies.

Price Erin P EP Huygens Flavia F Giffard Philip M PM

Applied and environmental microbiology 20060922 12

The aim of this investigation was to exploit the vast comparative data generated by comparative genome hybridization (CGH) studies of Campylobacter jejuni in developing a genotyping method. We examined genes in C. jejuni that exhibit binary status (present or absent between strains) within known plasticity regions, in order to identify a minimal subset of gene targets that provide high-resolution genetic fingerprints. Using CGH data from three studies as input, binary gene sets were identified w ...[more]

PMID: 16997982

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Project description:Conventional detection and confirmation methods for Campylobacter jejuni are lengthy and tedious. A rapid hybridization protocol in which a 1,475-bp chromogen-labelled DNA probe (pDT1720) and Campylobacter strains filtered and grown on 0.22-micron-pore-size hydrophobic grid membrane filters (HGMFs) are used was developed. Among the environmental and clinical isolates of C. jejuni, Campylobacter coli, Campylobacter jejuni subsp. doylei, Campylobacter lari, and Arcobacter nitrofigilis and a panel of 310 unrelated bacterial strains tested, only C. jejuni and C. jejuni subsp. doylei isolates hybridized with the probe under stringent conditions. The specificity of the probe was confirmed when the protocol was applied to spiked skim milk and chicken rinse samples. Based on the nucleotide sequence of pDT1720, a pair of oligonucleotide primers was designed for PCR amplification of DNA from Campylobacter spp. and other food pathogens grown overnight in selective Mueller-Hinton broth with cefoperazone and growth supplements. All C. jejuni strains tested, including DNase-producing strains and C. jejuni subsp. doylei, produced a specific 402-bp amplicon, as confirmed by restriction and Southern blot analysis. The detection range of the assay was as low as 3 CFU per PCR to as high as 10(5) CFU per PCR for pure cultures. Overnight enrichment of chicken rinse samples spiked initially with as little as approximately 10 CFU/ml produced amplicons after the PCR. No amplicon was detected with any of the other bacterial strains tested or from the chicken background microflora. Since C. jejuni is responsible for 99% of Campylobacter contamination in poultry, PCR and HGMF hybridization were performed on naturally contaminated chicken rinse samples, and the results were compared with the results of conventional cultural isolation on Preston agar. All samples confirmed to be culture positive for C. jejuni were also identified by DNA hybridization and PCR amplification, thus confirming that these DNA-based technologies are suitable alternatives to time-consuming conventional detection methods. DNA hybridization, besides being sensitive, also has the potential to be used in direct enumeration of C. jejuni organisms in chicken samples.

Dataset Information

Fingerprinting of Campylobacter jejuni by using resolution-optimized binary gene targets derived from comparative genome hybridization studies.

Publications

Fingerprinting of Campylobacter jejuni by using resolution-optimized binary gene targets derived from comparative genome hybridization studies.

Similar Datasets

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