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Inter‐laboratory evaluation of three flagellin PCR/RFLP methods for typing Campylobacter jejuni and C. coli : the CAMPYNET experience
Author(s) -
Harrington C.S.,
Moran L.,
Ridley A.M.,
Newell D.G.,
Madden R.H.
Publication year - 2003
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1046/j.1365-2672.2003.02101.x
Subject(s) - flagellin , typing , campylobacter jejuni , biology , amplicon , multiplex , restriction enzyme , restriction fragment length polymorphism , multiplex polymerase chain reaction , polymerase chain reaction , subtyping , microbiology and biotechnology , genetics , gene , bacteria , computer science , programming language
Aims: To compare typeability, discriminatory ability, and inter‐laboratory reproducibility of three flagellin PCR/RFLP ( fla typing) methods previously described for Campylobacter . Methods and Results: The sample set ( n = 100) was diverse, including both C. jejuni ( n = 85) and C. coli ( n = 15). Two of the three fla A typing methods amplified fla A alone, whereas one, a multiplex assay, amplified fla B in addition to fla A. Dde I restriction enzyme was employed for all methods, but Hin fI was also investigated. 98–100% typeability was obtained for fla A‐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 fla A over fla B. More Dde I types were generated using a longer fla A PCR amplicon, whilst additional use of Hin fI increased the number of types by ca 25%. Inter‐laboratory reproducibility for both fla A‐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 fla A gene and Dde I digestion, as an appropriate method on which to standardize. 100% inter‐laboratory reproducibility was demonstrated using that method. Significance and Impact of the Study: This work should facilitate progress towards inter‐laboratory standardization of fla typing.