Flagellin gene typing of Campylobacter jejuni by restriction fragment length polymorphism analysis

Abstract

We developed and studied a molecular typing approach for Campylobacter spp. with restriction fragment length polymorphism (RFLP) analysis of the flagellin geneflaA in C. jejuni. Using polymerase chain reaction, we amplified theJfaA gene from strains comprising different HL:O serotypes by using a primer set directed at the conserved 5' and 3'flaA gene sequence to generate a 1.7-kb amplicon. The amplicon was further digested with the restriction enzyme DdeI, and the fragments generated were analyzed by agarose gel electrophoresis. In 43 non-outbreak strains of six common HL serotypes (HL 1, 2, 4, 5, 9, and 36) in the United States, 18 RFLP patterns were observed. In U.S. outbreak strains previously studied by 10 other typing methods, faA typing correlated with the HL serotype within each outbreak, and six additionalflaA types were identified. Our results suggest that RFLP analysis of theflaA gene from Campylobacter spp. has sufficient discrimination to be useful as a practical typing method for clinical and epidemiologic investigations.

Restriction fragment length polymorphism analysis of the flaA gene of Campylobacter jejuni for subtyping human, animal and poultry isolates

“…We simpli¢ed protocols for preparing PCR template DNA from both DNAse +ve and 3ve strains of C. jejuni, and when combined with EcoRI/PstI RFLP analysis, the approach provided a fast (within 24 h) method of £aA-pro¢ling. Our results support those of Nachamkin et al [9,25] in showing that £aA amplicons can be obtained reproducibly from C. jejuni without the need for time-consuming DNA puri¢cation procedures. This technical simpli¢cation enables PCR to be done readily from just a few bacterial colonies or, if necessary, directly from preserved material without the need for culture.…”

Influence of temperature, oxygen and bacterial strain identity on the association of Campylobacter jejuni with Acanthamoeba castellanii

“…All isolates were identified at the species level using PCR as described by Vandamme et al (1997). Isolates were further characterized by flagellin gene A PCR/restriction fragment length polymorphism (flaA typing) using the restriction enzyme DdeI (Promega, Madison, WI) (Nachamkin et al, 1993) and pulsed-field gel electrophoresis (PFGE) using the restriction enzyme SmaI (Invitrogen, Paisley, UK) (Ribot et al, 2001). The presence of the invasion-related pldA gene and the pathogenic genes cdtA, cdtB and cdtC, responsible for cytolethal distending toxin (CDT) production, in the five C. jejuni strains was also determined as described by Hickey et al (2000) and Datta et al (2003).…”

Prevalence and strain diversity of thermophilic campylobacters in cattle, sheep and swine farms

“…DNA extracted as above was subjected to PCR amplification of a 1725 bp fragment of the flagellin flaA gene using the forward primer described by Fitzgerald et al (2001) and the reverse primer as reported by Nachamkin et al (1993). The 25 ll reactions were subjected to the following cycling conditions: 4 min at 94°C and 35 cycles of 15 s at 94°C, 45 s at 45°C and 1 min 45 s at 72°C.…”