| Summary: | <p>Abstract</p> <p>Background</p> <p>The human bacterial pathogen <it>Campylobacter jejuni </it>contains two subspecies: <it>C. jejuni </it>subsp. <it>jejuni </it>(<it>Cjj</it>) and <it>C. jejuni </it>subsp. <it>doylei </it>(<it>Cjd</it>). Although <it>Cjd </it>strains are isolated infrequently in many parts of the world, they are obtained primarily from human clinical samples and result in an unusual clinical symptomatology in that, in addition to gastroenteritis, they are associated often with bacteremia. In this study, we describe a novel multiplex PCR method, based on the nitrate reductase (<it>nap</it>) locus, that can be used to unambiguously subspeciate <it>C. jejuni </it>isolates.</p> <p>Results</p> <p>Internal and flanking <it>napA </it>and <it>napB </it>primer sets were designed, based on existing <it>C. jejuni </it>and <it>Campylobacter coli </it>genome sequences to create two multiplex PCR primer sets, <it>nap </it>mpx1 and <it>nap </it>mpx2. Genomic DNA from 161 <it>C. jejuni </it>subsp. <it>jejuni </it>(<it>Cjj</it>) and 27 <it>C. jejuni </it>subsp. <it>doylei </it>(<it>Cjd</it>) strains were amplified with these multiplex primer sets. The <it>Cjd </it>strains could be distinguished clearly from the <it>Cjj </it>strains using either <it>nap </it>mpx1 or mpx2. In addition, combination of either <it>nap </it>multiplex method with an existing <it>lpxA </it>speciation multiplex method resulted in the unambiguous and simultaneous speciation and subspeciation of the thermophilic Campylobacters. The <it>Cjd nap </it>amplicons were also sequenced: all <it>Cjd </it>strains tested contained identical 2761 bp deletions in <it>napA </it>and several <it>Cjd </it>strains contained deletions in <it>napB</it>.</p> <p>Conclusion</p> <p>The <it>nap </it>multiplex PCR primer sets are robust and give a 100% discrimination of <it>C. jejuni </it>subspecies. The ability to rapidly subspeciate <it>C. jejuni </it>as well as speciate thermophilic <it>Campylobacter </it>species, most of which are pathogenic in humans, in a single amplification will be of value to clinical laboratories in strain identification and the determination of the environmental source of campylobacterioses caused by <it>Cjd</it>. Finally, the sequences of the <it>Cjd napA </it>and <it>napB </it>loci suggest that <it>Cjd </it>strains arose from a common ancestor, providing clues as to the potential evolutionary origin of <it>Cjd</it>.</p>
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