The extent of sequence identity among clones derived from monomorphic and polymorphic AFLPTM polymorphism bands was quantified. A total of 79 fragments from a monomorphic band of 273 bp and 48 fragments from a polymorphic band of 159 bp, isolated from individuals belonging to different populations, varieties, and species of Echinacea, were cloned and sequenced. The monomorphic fragments exhibited above 90% sequence identity among clones within samples. Sequence identity within variety ranged from 82.78% to 94.87% and within species from 75.82% to 98.9% and was 57.97% in the genus. The polymorphic fragments exhibited much less sequence identity. In some instances, even two clones from the same fragment were different in their size and sequence. Within sample, clone sequence identity ranged from 100% to 51.57%, within variety from 33.33% to 100% in one variety, and from 23.66% to 45% within species and was as low as 1.25% within the genus. In addition, sequences of the same size were aligned to verify the nature of their sequence dissimilarity/similarity. Within each size group, identical sequences were found across species and varieties. In general, comigrating bands cannot be considered homologous. Thus, the use of AFLPTM band data for comparative studies is appropriate only if the results emanating from such analyses are considered as approximations and are interpreted as phenotypic but not genotypic.

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The goal of this study was to evaluate the suitability of the fimA gene amplification by PCR as a specific method for detection of Salmonella strains. Salmonella typhimurium and other pathogenic members of the family Enterobacteriaceae produce morphologically and antigenically related, thin, aggregative, type 1 fimbriae. A single gene, fimA, encodes the major fimbrial unit. In order to obtain higher specificity, we have selected a series of primers internal to the fimA gene sequence and have developed a PCR method for detecting Salmonella strains. A collection of 376 strains of Salmonella comprising over 80 serovars, isolated from animals and humans in Canada, have been used to evaluate this PCR method. Forty non-Salmonella strains were also tested by the same procedure. Cultures were screened by inoculating a single colony of bacteria directly into a PCR mixture containing a pair of primers specific for the fimA gene. The specific PCR product is an 85-bp fragment which was visualized by polyacrylamide gel electrophoresis and ethidium bromide staining. All Salmonella strains gave positive results by the PCR. Feed and milk samples contaminated by Salmonella strains were also detected by this procedure. The detection of all Salmonella strains tested and the failure to amplify the fragment from non-Salmonella strains confirm that the fimA gene contains sequences unique to Salmonella strains and demonstrate that this gene is a suitable PCR target for detection of Salmonella strains in food samples.

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