Inter-laboratory evaluation of three flagellin PCR/RFLP methods for typing Campylobacter jejuni and C. coli: the CAMPYNET experience

Harrington, Clare S.; Moran, L.; Ridley, Anne; Newell, Diane G.; Madden, Robert H.

doi:10.1046/j.1365-2672.2003.02101.x

Cited by 54 publications

(46 citation statements)

References 29 publications

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“…PCR amplification was performed with a Ready-to-Go system (GE Healthcare, Little Chalfont, UK). RFLP-flaA with the restriction enzyme DdeI (Roche, Madrid, Spain) was performed following the Campynet protocol (Harrington et al, 2003).…”

Section: Methodsmentioning

confidence: 99%

Drinking water as the source ofCampylobacter coliinfection in grandparent heavy breeders

Pérez-Boto

García-Peña²,

Abad-Moreno³

et al. 2010

Avian Pathology

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Section: Methodsmentioning

confidence: 99%

Drinking water as the source ofCampylobacter coliinfection in grandparent heavy breeders

Pérez-Boto

García-Peña²,

Abad-Moreno³

et al. 2010

Avian Pathology

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“…These observations are largely consistent with data from large-scale comparative genomic studies that were compared to the C. jejuni NCTC11168 genome sequence (26) showing that significant regions of the C. jejuni genome are stable (32) but that as much as 21% of genes in NCTC11168 may be dispensable (8). More recently, an interlaboratory evaluation of three flagellin PCR/RFLP methods for typing C. jejuni and C. coli showed that analysis of the full flaA gene using the restriction endonuclease DdeI was an appropriate method for standardization, as demonstrated by 100% interlaboratory reproducibility (12). Collectively, these studies suggest that RFLP-flaA is a discriminatory, reliable, and relatively inexpensive typing tool for Campylobacter.…”

mentioning

confidence: 99%

Clonal Complexes of Campylobacter jejuni Identified by Multilocus Sequence Typing Are Reliably Predicted by Restriction Fragment Length Polymorphism Analyses of the flaA Gene

et al. 2007

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Multilocus sequence typing (MLST) has provided important new insights into the population structure ofCampylobacter jejuni and is rapidly becoming the gold standard for typing this species. However, the methodology is comparatively costly and slow to perform for the routine surveillance testing of large numbers of isolates required by public health laboratories. Restriction fragment length polymorphism analysis of the flaA gene (RFLP-flaA) and sequencing of the variable region in the fla locus (SVR-fla) were compared to MLST to determine if a low cost alternative could be found that reliably predicts clonal lineage (as determined by MLST). An isolate of C. jejuni from each of 153 patients from New South Wales, Australia, collected sequentially over a period of 30 months from 1999 to 2001 and comprising 40 sequence types (ST) from 15 clonal complexes (CC) was examined. Of 15 CC, 12 were represented by more than one isolate and a predominant RFLP-flaA type was found for 10 (83%). Of these, seven (70%) correctly predicted the predominant MLST CC with a probability of >0.8. Of 40 STs detected, 19 were reported for the first time, 9 of which were represented by more than one isolate. Eight of these were represented by a single RFLP-flaA type. Only two of eight major SVR-fla types were able to predict CC with a probability of >0.8, indicating that flaA-RFLP is a more reliable predictor of CC than SVR-fla and thus offers an alternative to MLST for use in routine surveillance.

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“…For Campylobacter spp., amplified fragment length polymorphism methods are at least as discriminatory as multilocus sequence typing (MLST) and may be as discriminatory as PFGE (13,32); they are certainly less labor-intensive than PFGE (3). Flagellin locus restriction fragment length polymorphism (fla-RFLP) methods have been developed and have proved both epidemiologically relevant and very useful for strain discrimination in an outbreak analysis (14,21,22,23,25). Analysis of the flagellin short variable region (fla-SVR) yielded results very similar to those obtained by sequencing of the entire gene (21).…”

mentioning

confidence: 99%

Use of the Oxford Multilocus Sequence Typing Protocol and Sequencing of the Flagellin Short Variable Region To Characterize Isolates from a Large Outbreak of Waterborne Campylobacter sp. Strains in Walkerton, Ontario, Canada

et al. 2005

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The Walkerton (Ontario, Canada) outbreak of waterborne Escherichia coli O157:H7 and Campylobacter jejuni was quite limited in both space and time, making it a good model for exploring the utility of different typing and subtyping methods for the characterization of relationships among isolates of these organisms. We have extended previous work with these organisms through analysis by the Oxford multilocus sequence typing (MLST) and the flagellin short variable region (fla-SVR) sequencing methods. Additional isolates not epidemiologically related to the Walkerton outbreak have also been included. Both sequencing methods identified and differentiated between Walkerton outbreak strains 1 and 2. When these strains were compared with isolates that were not part of the outbreak, the information produced by the fla-SVR method more often correlated with epidemiological findings than that produced by MLST, though both methods were required for optimal discrimination. The MLST data were more relevant in terms of the overall population structure of the organisms. Both mutation and recombination appeared to be responsible for generating diversity among the isolates tested.

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Inter-laboratory evaluation of three flagellin PCR/RFLP methods for typing Campylobacter jejuni and C. coli: the CAMPYNET experience

Abstract: This work should facilitate progress towards inter-laboratory standardization of fla typing.

Cited by 54 publications

References 29 publications

Drinking water as the source ofCampylobacter coliinfection in grandparent heavy breeders

Drinking water as the source ofCampylobacter coliinfection in grandparent heavy breeders

Clonal Complexes of Campylobacter jejuni Identified by Multilocus Sequence Typing Are Reliably Predicted by Restriction Fragment Length Polymorphism Analyses of the flaA Gene

Use of the Oxford Multilocus Sequence Typing Protocol and Sequencing of the Flagellin Short Variable Region To Characterize Isolates from a Large Outbreak of Waterborne Campylobacter sp. Strains in Walkerton, Ontario, Canada

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