For an empirical demonstration of our algorithm, we tested a multiplex set of 16 short, 10-nt primers designed for the Poxviridae viral family against commercially purified vaccinia virus extracts. The primer sequences are provided in Table 4 along with the predicted single amplicon for vaccinia Lister strain. We chose the Poxviridae family multiplex for these first empirical demonstrations because extracted viral nucleic acid was readily available for experiments. All primers were purchased from Integrated DNA Technologies (Coralville, IA, USA) and resuspended to 100 mM stock solutions in TE buffer (pH 8.0, Teknova, Hollister, CA, USA). Working solutions containing equimolar concentrations of each of the 16 primers were used in all experiments. Purified, quantitated vaccinia Lister strain DNA was purchased at a concentration of 1.3 Â 10 4 copies/ml in nuclease-free water from Advanced Biotechnologies, Inc. (Columbia, MD, USA). All PCR experiments were prepared using the Superscript III RT-PCR kit from Invitrogen (Carlsbad, CA, USA). We selected the RT-PCR kit in order to establish a protocol that could later be readily applied to additional multiplex viral family reactions with viral DNA and/or RNA. Each 25 ml reaction contained 1 Â SSIII buffer, 1 U of SSIII RT/ Taq enzyme, 4.8 mM MgSO 4 , 0.1 mM each primer, and a viral template mass of 2.7 pg ($10 4 copies). Tests were performed in triplicate and corresponding negative controls were run under identical conditions except that viral template was replaced with nuclease-free water (Ambion, Austin, TX, USA). All reactions were thermocycled on the Bio-Rad DNA Engine (Hercules, CA, USA) as follows: one cycle of 2 min at 94 C; 40 cycles of 15 s at 94 C, 30 s at 43.9 C, 1 min at 68 C; one cycle of 5 min at 68 C. In line with our goal to create a protocol applicable to both DNA and/or RNA templates, we verified that inclusion of an RT step (one cycle of 30 min at 45 C) does not alter the outcome of the subsequent PCR when working with DNA template (data not shown). As such, for all Poxviridae 16-plex results reported here, an RT step was not used. Another goal was to establish reaction conditions that can be applied to any multiplex viral primer set without the need for re-optimization, assuming the primer set is designed with the same general parameter contraints (T m s, etc.) used to compute the 16-plex presented here. As such, we first optimized master mix conditions, where it was determined that a 4.8 mM MgSO 4 concentration resulted in most optimal amplification. Similarly, we determined the optimal annealing temperature based on results from annealing temperature gradient experiments. A more detailed discussion on the optimization of reaction conditions for amplification with our short primer viral multiplexes is the subject of a separate paper in preparation (Hiddessen and co-workers).