Inter-laboratory evaluation of three flagellin PCR/RFLP methods for typing Campylobacter jejuni and C. coli: the CAMPYNET experience.
OBJECTIVE
To compare typeability, discriminatory ability, and inter-laboratory reproducibility of three flagellin PCR/RFLP (fla typing) methods previously described for Campylobacter.


RESULTS
The sample set (n = 100) was diverse, including both C. jejuni (n = 85) and C. coli (n = 15). Two of the three flaA typing methods amplified flaA alone, whereas one, a multiplex assay, amplified flaB in addition to flaA. DdeI restriction enzyme was employed for all methods, but HinfI was also investigated. 98-100% typeability was obtained for flaA-based methods, but only 93% for the multiplex assay, due to inconsistent amplification of a non-specific product. In addition, there appeared to be selective amplification of flaA over flaB. More DdeI types were generated using a longer flaA PCR amplicon, whilst additional use of HinfI increased the number of types by ca 25%. Inter-laboratory reproducibility for both flaA-based methods was defined at 100%.


CONCLUSIONS
Fla typing requires standardization with respect to PCR primers and restriction enzymes. This study identified an assay, employing the full flaA gene and DdeI digestion, as an appropriate method on which to standardize. 100% inter-laboratory reproducibility was demonstrated using that method.


CONCLUSIONS
This work should facilitate progress towards inter-laboratory standardization of fla typing.
Harrington
C S
CS
The Queen's University of Belfast, Belfast, UK. csh@vetinst.dk
Moran
L
L
Ridley
A M
AM
Newell
D G
DG
Madden
R H
RH
D000818:Animals; D015373:Bacterial Typing Techniques / Q000379:methods*; D017000:Campylobacter coli / Q000145:classification*; D016123:Campylobacter jejuni / Q000145:classification*; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000235:genetics; D005408:Flagellin / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D015203:Reproducibility of Results
D004269:DNA, Bacterial; D005408:Flagellin