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Appl. Environ. Microbiol. doi:10.1128/AEM.02269-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Development of a Strain-Specific Molecular Method for Quantitating Individual Campylobacter Strains in Mixed Populations

University of Bristol, Langford, Bristol, BS40 5DU, UK; Veterinary Laboratories Agency, Addlestone, Surrey, KT15 3NB, UK; Molecular Microbiology and Genomics Consultants, Tannenstrasse 7, 55576 Zotzenheim, Germany

^* To whom correspondence should be addressed. Email: Karen.Elvers{at}bristol.ac.uk.

	Abstract

The identification of sites resulting in cross-contamination of poultry flocks in the abattoir, and the survival and persistence of Campylobacters at these sites is essential for the development of intervention strategies aimed at reducing the microbial burden on poultry at retail. A novel molecular-based method, using strain- and genus-specific oligonucleotide probes, has been developed to detect and enumerate specific campylobacter strains in mixed populations. Strain-specific oligonucleotide probes were designed against the short variable regions (SVR) of the flaA gene from individual Campylobacter jejuni strains. A 16S rRNA Campylobacter genus-specific probe was also used. Both probes were used to investigate populations of campylobacters by colony lift hybridisation. The specificity and proof of principle of the method was tested using strains with closely related SVR sequences and with mixtures of these strains. Colony lifts of campylobacters were hybridised sequentially with up to two labelled strain-specific probes followed by the generic 16S rRNA probe. SVR probes were highly specific, differentiating down to one nucleotide in the target sequence, and sufficiently sensitive to detect colonies of a single strain in a mixed population. The 16S rRNA probe detected all Campylobacter sp. tested but not closely related species such as Arcobacter skirrowi or Helicobacter pullorum. Preliminary field studies have demonstrated the application of this technique to target strains isolated from poultry transport crate wash tank water. This method is quantitative, sensitive and highly specific and allows the identification and enumeration of selected-strains among the total number of Campylobacters in environmental samples.