Table 1.
Amplicon lengths, thermoprofiles and probe modifications of the TaqMan qPCR and RT-qPCR assays included in the study.
Fig 1.
Effect of the SYTO 82 dye on the FAM-TaqMan assay.
(A) The mean FAM d(RFU) data of the S82 gradient, ranges from 0 to 10μM, in 1μM increments, were shown for the IAV assay (Table 1) performed as a qPCR. Each amplification curve represents an average of three TaqMan reaction replicates per S82 concentration for a fixed initial synthetic DNA template amount of 1e4 copies/μl. The linear regression lines drawn across the exponential region of each amplification curve are dashed, and the region used for regression line construction is highlighted in blue. The slope values k were designated for the two S82 concentration extremities. For additional details, please refer to Table A and Fig BA in S2 File. (B), the Cq plot was constructed by plotting the Cq values of the IAV (blue) and FMDV (brown) assays against the S82 concentration. The data obtained from the FAM and VIC channels were designated with filled marks and solid lines or empty marks and dashed lines, respectively. (C-F) illustrate the FAM and VIC channel amplification curves of the IAV and FMDV assays in logarithmic views for one replicate series.
Fig 2.
Effect of the SYTO 82 dye on the melting peak profiles.
(A)The mean melting curve derivative data were shown for the IAV assay (Table 1) performed as a qPCR. Each melting peak represents an average of three replicates per S82 concentration with a fixed initial synthetic DNA template amount of 1e4 copies/μl. The S82 gradient, ranges from 1 to 10μM, in 1μM increments, was designated with a blue arrow. For each peak, the left tail local minimum and maximum values used for the estimation of the peak height H and proportionality, %H, were designated with blue spots. (B), the melting peak height and proportionality values plotted against the S82 concentration. The melting peak heights for the FMDV (brown, Fig D in S2 File) and IAV assays (blue) were designated with dashed lines and open symbols and related to the primary y axis. The secondary y axis indicates the %H values visualized by filled symbols and solid lines. For additional details, please refer to Table A in S2 File.
Fig 3.
Evaluation of the MeltMan assay.
The concentration gradient of the FMDV qPCR (Table 1) from 1e2 to 1e6 synthetic DNA template copies/μl in three replicates (A) and the calibration curves (B). For the amplification and calibration curves of the IAV assay please refer to Figs EA and EB in S2 File. The results for no S82 and S82 subsets were highlighted in grey and brown, respectively. For clarity, certain curves were visualized as dashed. (C) the d(RFU) FAM data. (D and E) the ΔCq and %H plots for the FMDV (brown), and IAV (blue) assays, respectively. The data are corresponding with Table C in S2 File.
Fig 4.
Sensitivity of the MeltMan reaction system.
Seven TaqMan assays: Chicken DNA, EHV-1, TBEV, OvHV-2, IAV, EAV, and Celery DNA (Table 1) were analysed by using diluted field specimens to reach Cq values of ≥30. The assays were prepared in two subsets, no S82 (grey) and S82 (red), with ten replicates per subset. The graph represents the Cq values obtained for each particular replicate. The data are corresponding with Fig F and Table D in S2 File.
Fig 5.
Compatibility of the S82 dye with various TaqMan assays.
The qPCR (A) and RT-qPCR ΔCq scatter plots (B) show the distribution of the ΔCq values per each S82 and no S82 paired reaction obtained by four different baseline approaches. The frequencies of the particular ΔCq values are provided in Figs GC and GD in S2 File.