Publication
Differentiation of Campylobacter coli, Campylobacter jejuni, Campylobacter lari, and Campylobacter upsaliensis by a multiplex PCR developed from the nucleotide sequence of the lipid A gene lpxA
Publisher:
American Society for Microbiology
Date:
12-2004
DOI:
10.1128/JCM.42.12.5549-5557.2004
Abstract: We describe a multiplex PCR assay to identify and discriminate between isolates of C ylobacter coli , C ylobacter jejuni , C ylobacter lari , and C ylobacter upsaliensis . The C. jejuni isolate F38011 lpxA gene, encoding a UDP- N -acetylglucosamine acyltransferase, was identified by sequence analysis of an expression plasmid that restored wild-type lipopolysaccharide levels in Escherichia coli strain SM105 [ lpxA (Ts)]. With oligonucleotide primers developed to the C. jejuni lpxA gene, nearly full-length lpxA licons were lified from an additional 11 isolates of C. jejuni , 20 isolates of C. coli , 16 isolates of C. lari , and five isolates of C. upsaliensis . The nucleotide sequence of each licon was determined, and sequence alignment revealed a high level of species discrimination. Oligonucleotide primers were constructed to exploit species differences, and a multiplex PCR assay was developed to positively identify isolates of C. coli , C. jejuni , C. lari , and C. upsaliensis. We characterized an additional set of 41 thermotolerant isolates by partial nucleotide sequence analysis to further demonstrate the uniqueness of each species-specific region. The multiplex PCR assay was validated with 105 genetically defined isolates of C. coli , C. jejuni , C. lari , and C. upsaliensis , 34 strains representing 12 additional C ylobacter species, and 24 strains representing 19 non- C ylobacter species. Application of the multiplex PCR method to whole-cell lysates obtained from 108 clinical and environmental thermotolerant C ylobacter isolates resulted in 100% correlation with biochemical typing methods.