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MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay.

Nagy A, Černíková L, Vitásková E, Křivda V, Dán Á, Dirbáková Z, Jiřincová H, Procházka B, Sedlák K, Havlíčková M - PLoS ONE (2016)

Bottom Line: We proved that the S82 is fully compatible with the FAM-TaqMan system.Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples.These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Methods, State Veterinary Institute Prague, Prague, Czech Republic.

ABSTRACT
In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence.

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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.
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pone.0151204.g003: 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.

Mentions: As seen in Fig 3A (Fig EA in S2 File), the early exponential phases of the corresponding amplification curves for the subsets tested were tightly overlapped across the entire dilution range. This resulted in highly similar Cq values in both assays and was also reflected in the perfectly parallel and overlapping standard curves with almost identical efficiency, slope, and R2 values (Fig 3B; Fig EB and Table C in S2 File).


MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay.

Nagy A, Černíková L, Vitásková E, Křivda V, Dán Á, Dirbáková Z, Jiřincová H, Procházka B, Sedlák K, Havlíčková M - PLoS ONE (2016)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4816343&req=5

pone.0151204.g003: 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.
Mentions: As seen in Fig 3A (Fig EA in S2 File), the early exponential phases of the corresponding amplification curves for the subsets tested were tightly overlapped across the entire dilution range. This resulted in highly similar Cq values in both assays and was also reflected in the perfectly parallel and overlapping standard curves with almost identical efficiency, slope, and R2 values (Fig 3B; Fig EB and Table C in S2 File).

Bottom Line: We proved that the S82 is fully compatible with the FAM-TaqMan system.Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples.These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Methods, State Veterinary Institute Prague, Prague, Czech Republic.

ABSTRACT
In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence.

Show MeSH