Table 1.
Assay sequences and measures of specificity.
Table 2.
Quantification of stock solutions for assay sensitivity experiments.
Table 3.
Assay BRK2 sensitivity results.
Figure 1.
Impact of base pair mismatches on fluorescence.
The relationship between end-point fluorescence (measured as proportional S/N; the fluorescence at cycle 40 divided by the fluorescence at cycle 1 as a proportion of a positive control) and base pair mismatches in the primer regions (A), probe region (B), and primers and probe regions combined (C). End-point fluorescence decreases as the number of primer base pair mismatches increases.
Figure 2.
Non-target template competition in the brook trout assays.
Part A shows the amplification curves of BRK1 when using DNA solutions from brook trout, bull trout, and brook trout mixed into bull trout at 1∶10 and 1∶100 dilutions. Part B shows the amplification curves of BRK1 when using DNA solutions from brook trout, lake trout, and brook trout mixed into lake trout at 1∶10 and 1∶100 dilutions. Part C shows the amplification curves of BRK2 when using DNA solutions from brook trout, bull trout, and brook trout mixed into bull trout at 1∶10 and 1∶100 dilutions. Part D shows the amplification curves of BRK2 when using DNA solutions from brook trout, lake trout, and brook trout mixed into lake trout at 1∶10 and 1∶100 dilutions. Assay BRK1 has a single primer-base-pair mismatch and three probe-base-pair mismatches with bull trout, but produces an ambiguous signal when brook trout represent a small proportion of the sample. BRK2 has nine primer base pair mismatches and a single probe base pair mismatch with bull trout, and is still sensitive even when brook trout represents a small proportion of the sample. The presence of lake trout DNA does not appear to influence the sensitivity of either assay.
Figure 3.
Non-target template competition in the bull trout assay.
Part A shows the amplification curves of BUT1 when using DNA solutions from bull trout, lake trout, and bull trout mixed into lake trout at 1∶10 and 1∶100 dilutions. Part B shows the amplification curves of BUT1 when using DNA solutions from bull trout, brook trout, and bull trout mixed into brook trout at 1∶10 and 1∶100 dilutions. The assay primers have nine and five primer base pair mismatches with brook trout and lake trout respectively. Four of the mismatches with brook trout are within five base pairs of the 3′ primer ends, but none of the mismatches with lake trout are near the 3′ ends. The assay is not influenced by the presence of brook trout, but produces an ambiguous signal when bull trout represents a small portion of the sample.