Improved repetitive-element PCR fingerprinting of Salmonella enterica with the use of extremely elevated annealing temperatures.

Bacterial strain typing at the subspecific level is an essential tool for contemporary public health and hospital infection control efforts (8, 18, 27, 33), as well as for basic research involving the molecular epidemiology and evolutionary biology of pathogenic bacteria (2, 6, 10, 21–24). Traditional subspecific typing methods include serotyping, phage typing, biotyping, plasmid profiling, multilocus enzyme electrophoresis, conventional restriction endonuclease analysis, ribotyping, and pulsed-field gel electrophoresis (PFGE). Their strengths notwithstanding, all of these methods have one or more significant drawbacks, including being slow or cumbersome; requiring highly specialized equipment, skills, and/or reagents; relying on variable or unstable traits; and yielding uninterpretable results for some strains (8, 18, 23, 33). PCR-based fingerprinting is a simple, rapid, and broadly applicable typing method that is potentially available to any laboratory with PCR capability. Fingerprints are generated using either arbitrary primers (random amplified polymorphic DNA [35], arbitrarily primed PCR [34], or DNA amplification fingerprinting [5]) or repetitive-element-based primers (rep-PCR) (32). PCR fingerprinting has been reported to be useful in a variety of infection control and molecular epidemiological applications involving diverse bacterial types (11–13, 25, 27, 29, 31, 36). However, concerns have been raised regarding the irreproducibility of PCR-generated fingerprints (1, 20, 27; E. M. Jutras, P. Rochelle, R. de Leon, M. Stewart, and R. Wolfe, Abstr. 98th Gen. Meet. Am. Soc. Microbiol., abstr. Q-109, p. 439, 1998). Claims of reproducibility from proponents of PCR fingerprinting generally have not been supported with specific data. On the contrary, reports of manipulations which ostensibly improve the reproducibility of PCR fingerprinting (7, 9, 13, 32; G. Lisby, D. L. Baggesen, and U. Skibsted, Abstr. 98th Gen. Meet. Am. Soc. Microbiol., abstr. L-6, p. 354, 1998) suggest that irreproducibility is a greater problem with this method than is generally acknowledged. Although our preliminary experience with rep-PCR typing of E. coli (14–16) and Salmonella enterica (J. R. Johnson, unpublished data) confirmed the method's speed and simplicity, we also found that with the published cycling conditions (32) the assay's reproducibility and strain discrimination on a day-to-day basis were inadequate, even with the use of kit-purified genomic DNA instead of boiled lysates and commercial PCR beads instead of hand-compounded master mixes (M. Saluta, W.-T. E. Ting, M. Koonge, and C. Tseng, Abstr. 98th Gen. Meet. Am. Soc. Microbiol., abstr. H-124, p. 297, 1998). The comparatively high GC content of the ERIC and BOXA1R primers used in rep-PCR (32) suggested to us that at the recommended annealing temperature (52°C) these primers might bind to and initiate DNA synthesis from partially mismatched recognition sites. Such mismatched annealing would be expected to be unstable and highly temperature dependent and hence sensitive to small temperature shifts as might occur from run to run or from day to day on a given thermal cycler or between cyclers. We hypothesized that the use of higher annealing temperatures would yield more specific (and hence more temperature-stable) priming, resulting in greater day-to-day and cycler-to-cycler reproducibility of fingerprints. (This is similar to the principle underlying “touchdown” [TD] cycling, in which to increase the specificity of primer binding in early cycles, annealing temperatures are initially set higher than the ultimate annealing temperature and then are decreased in a stepwise fashion with each cycle until the ultimate annealing temperature is reached [7, 9].) In the present study we sought to rigorously and quantitatively evaluate the impact of extremely elevated annealing temperatures on the reproducibility and discriminating power of rep-PCR fingerprints, using as the test substrate strains representing different serovars of S. enterica.