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Background  The human bacterial pathogen  Campylobacter jejuni  contains two subspecies:  C. jejuni  subsp.  jejuni  ( Cjj ) and  C. jejuni  subsp.  doylei  ( Cjd ). Although  Cjd  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 ( nap ) locus, that can be used to unambiguously subspeciate  C. jejuni  isolates.    Results  Internal and flanking  napA  and  napB  primer sets were designed, based on existing  C. jejuni  and  Campylobacter coli  genome sequences to create two multiplex PCR primer sets,  nap  mpx1 and  nap  mpx2. Genomic DNA from 161  C. jejuni  subsp.  jejuni  ( Cjj ) and 27  C. jejuni  subsp.  doylei  ( Cjd ) strains were amplified with these multiplex primer sets. The  Cjd  strains could be distinguished clearly from the  Cjj  strains using either  nap  mpx1 or mpx2. In addition, combination of either  nap  multiplex method with an existing  lpxA  speciation multiplex method resulted in the unambiguous and simultaneous speciation and subspeciation of the thermophilic Campylobacters. The  Cjd nap  amplicons were also sequenced: all  Cjd  strains tested contained identical 2761 bp deletions in  napA  and several  Cjd  strains contained deletions in  napB .    Conclusion  The  nap  multiplex PCR primer sets are robust and give a 100% discrimination of  C. jejuni  subspecies. The ability to rapidly subspeciate  C. jejuni  as well as speciate thermophilic  Campylobacter  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  Cjd . Finally, the sequences of the  Cjd napA  and  napB  loci suggest that  Cjd  strains arose from a common ancestor, providing clues as to the potential evolutionary origin of  Cjd .

bmc microbiology

Identification of genomic differences between Campylobacter jejuni subsp. jejuni and C. jejuni subsp. doylei at the nap locus leads to the development of a C. jejuni subspeciation multiplex PCR method