Genotyping primers for fully automated multilocus variable-number tandem repeat analysis of Escherichia coli O157:H7.

We recently developed a multilocus variable-number tandem repeat (TR) analysis (MLVA) assay for Escherichia coli O157:H7 (2). In this assay, we identified seven loci that, when used in combination, were able to identify E. coli O157:H7 outbreaks, discriminate among genetically diverse isolates, and discriminate among isolates that were found to be highly related by pulsed-field gel electrophoresis but not known to be associated with an outbreak. In our paper, we supplied primers for the seven loci (2). These primers successfully amplified the appropriate tandem repeat loci, and by sequencing each locus and counting the number of repeats we were able to assign an MLVA type for each isolate. We now have automated the process with new primers, using the approach described for Bacillus anthracis (1). Our fluorescently labeled primers have been designed to give each locus a discrete range on the sequencer, when pos-