Molecular discrimination betweenCampylobacter jejuni,Campylobacter coli,Campylobacter lariandCampylobacter upsaliensisby polymerase chain reaction based on a novel putative GTPase gene

Doorn, Leen–Jan van; Giesendorf, B. A. J.; Bax, R.; Zeijst, Bernard A.M. van der; Vandamme, Peter; Quint, Wim

doi:10.1006/mcpr.1997.0100

Cited by 22 publications

(17 citation statements)

References 26 publications

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“…The observations that the members of the species C. lari are phenotypically and genotypically diverse and that the species may comprise multiple taxa are in concordance with the findings presented in several other reports and highlight the fact that the taxonomy of C. lari is still in progress (4,10,11,12,32,37). Since C. jejuni, C. coli, and C. lari are significant pathogens and their differentiation is important when they are involved in clinical cases of infection, we suggest the use of recently described PCR assays for accurate discrimination and identification of the respective taxon (16,29,49,50). The identification of taxa to the subspecies level was possible for 14 of 15 strains of C. hyointestinalis.…”

Section: Discussionmentioning

confidence: 99%

Species-Specific Identification of Campylobacters by Partial 16S rRNA Gene Sequencing

Gorkiewicz

Feierl²,

Schober

et al. 2003

J Clin Microbiol

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Species-specific identification of campylobacters is problematic, primarily due to the absence of suitable biochemical assays and the existence of atypical strains. 16S rRNA gene (16S rDNA)-based identification of bacteria offers a possible alternative when phenotypic tests fail. Therefore, we evaluated the reliability of 16S rDNA sequencing for the species-specific identification of campylobacters. Sequence analyses were performed by using almost 94% of the complete 16S rRNA genes of 135 phenotypically characterized Campylobacter strains, including all known taxa of this genus. It was shown that 16S rDNA analysis enables specific identification of most Campylobacter species. The exception was a lack of discrimination among the taxa Campylobacter jejuni and C. coli and atypical C. lari strains, which shared identical or nearly identical 16S rDNA sequences. Subsequently, it was investigated whether partial 16S rDNA sequences are sufficient to determine species identity. Sequence alignments led to the identification of four 16S rDNA regions with high degrees of interspecies variation but with highly conserved sequence patterns within the respective species. A simple protocol based on the analysis of these sequence patterns was developed, which enabled the unambiguous identification of the majority of Campylobacter species. We recommend 16S rDNA sequence analysis as an effective, rapid procedure for the specific identification of campylobacters.Campylobacter species are important pathogens that cause a variety of diseases in humans and animals (26, 43). The most prominent members of these proteobacteria are the species Campylobacter coli and C. jejuni, the latter of which is considered the most common cause of acute bacterial enteritis worldwide (3). To date, the genus Campylobacter comprises 16 species, and among them several species other than C. jejuni and C. coli are becoming increasingly recognized as significant human pathogens (26). However, recovery and identification of these species require specialized preparatory procedures for specimens, such as filtration steps and selective incubation methods (e.g., the use of a hydrogen-enriched atmosphere) (26). Since most routine laboratories do not use these techniques, infections caused by these taxa are likely to be underdiagnosed (13,27). In addition, phenotypic tests have only a limited discriminatory potential for the distinctive identification of Campylobacter species. These pathogens are slowly growing, fastidious organisms and are considered biochemically unreactive. As a result, extensive identification schemes comprising up to 67 phenotypic features are used to correctly identify the entire spectrum of campylobacteria (40). Moreover, the phenotypic tests used in most routine laboratories lack standardization, although it is known that even minor parameters such as the inoculum size affect the results (39). The existence of biochemically atypical strains, which exhibit unusual phenotypic profiles, represents an additional challenge (36). In these cases no...

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Section: Discussionmentioning

confidence: 99%

Species-Specific Identification of Campylobacters by Partial 16S rRNA Gene Sequencing

Gorkiewicz

Feierl²,

Schober

et al. 2003

J Clin Microbiol

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“…The gene encoding the GTP-binding protein (29,30) and the glyA gene, which encodes serine hydroxymethyltransferase (1), can identify four Campylobacter species, but neither has yet been applied to clinical samples. We have previously described a PCR-ELISA with first-and second-round pangenus PCRs that eliminated the need for multiple single-species reactions and then achieved identification by probe hybridization (20).…”

Section: Discussionmentioning

confidence: 99%

Rapid Identification of Campylobacter spp. by Melting Peak Analysis of Biprobes in Real-Time PCR

et al. 2001

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We describe rapid PCR-biprobe identification of Campylobacter spp.. This is based on real-time PCR with product analysis in the same system. The assay identifies enteropathogenic campylobacters to the species level on the basis of their degree of hybridization to three 16S ribosomal DNA (rDNA) biprobes. First-round symmetric PCR is performed with genus-specific primers which selectively target and amplify a portion of the 16S rRNA gene common to all Campylobacter species. Second-round asymmetric PCR is performed in a LightCycler in the presence of one of three biprobes; the identity of an amplified DNA-biprobe duplex is established after determination of the species-specific melting peak temperature. The biprobe specificities were determined by testing 37 reference strains of Campylobacter, Helicobacter, and Arcobacter spp. and 59 Penner serotype reference strains of Campylobacter jejuni and C. coli. From the combination of melting peak profiles for each probe, an identification scheme was devised which accurately detected the five taxa pathogenic for humans (C. jejuni/C. coli, C. lari, C. upsaliensis, C. hyointestinalis, and C. fetus), as well as C. helveticus and C. lanienae. The assay was evaluated with 110 blind-tested field isolates; when the code was broken their previous phenotypic species identification was confirmed in every case. The PCR-biprobe assay also identified campylobacters directly from fecal DNA. PCR-biprobe testing of stools from 38 diarrheic subjects was 100% concordant with PCR-enzyme-linked immunosorbent assay identification (13, 20) and thus more sensitive than phenotypic identification following microaerobic culture.

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“…Oligodeoxynucleotide primers and PCR assays that distinguish between one or more combinations of the thermotolerant Campylobacter species have been described (1,4,5,9,11,13,16,21,48). The advantages of PCRbased assays are that they can be performed quickly and relatively cheaply (29).…”

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confidence: 99%

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

et al. 2004

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We describe a multiplex PCR assay to identify and discriminate between isolates of Campylobacter coli, Campylobacter jejuni, Campylobacter lari, and Campylobacter 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 amplicons were amplified 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 amplicon 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 Campylobacter species, and 24 strains representing 19 non-Campylobacter species. Application of the multiplex PCR method to whole-cell lysates obtained from 108 clinical and environmental thermotolerant Campylobacter isolates resulted in 100% correlation with biochemical typing methods.

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Molecular discrimination betweenCampylobacter jejuni,Campylobacter coli,Campylobacter lariandCampylobacter upsaliensisby polymerase chain reaction based on a novel putative GTPase gene

Cited by 22 publications

References 26 publications

Species-Specific Identification of Campylobacters by Partial 16S rRNA Gene Sequencing

Species-Specific Identification of Campylobacters by Partial 16S rRNA Gene Sequencing

Rapid Identification of Campylobacter spp. by Melting Peak Analysis of Biprobes in Real-Time PCR

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

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