Improved repetitive-element PCR fingerprinting of Salmonella enterica withthe use of extremely elevated annealing temperatures

Modified thermal cycling conditions were explored in an effort to improve t he reproducibility and resolving power of repetitive-element PCR (rep-PCR) fingerprinting. Assay performance was rigorously evaluated under standard a nd modified cycling conditions, using as a test set 12 strains putatively r epresenting 12 serovars of Salmonella enterica, For all three fingerprint t ypes (ERIC2, BOXA1R, and composite fingerprints), the use of extremely elev ated annealing temperatures plus an initial "touchdown" cycling routine yie lded significant improvements in day-to-day reproducibility and discriminat ing power despite the somewhat sparser appearance of the fingerprints. Modi fied cycling conditions markedly reduced the variability of fingerprints be tween cyclers, allowing fingerprints from different cyclers to be analyzed together without the degradation of assay performance that occurred with be tween-cycler analyses under standard cycling conditions. With modified cycl ing, composite fingerprints exhibited the lowest reproducibility but the hi ghest net discriminating power of the three Fingerprint types. rep-PCR fing erprints led to the discovery of a serotyping error involving one of the 12 test strains. These data demonstrate that modified cycling regimens that i ncorporate elevated annealing temperatures (with or without an initial touc hdown routine) may markedly improve the performance of rep-PCR fingerprinti ng as a bacterial typing tool.