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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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Related in: MedlinePlus

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.
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pone.0151204.g001: 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.

Mentions: The evaluation of the S82 gradient showed that increasing concentration of the dye had a remarkable suppressive effect on the FAM fluorescence curve (Fig 1A; Table A and Fig BA in S2 File). To quantify the observed changes, the slope values, k, of the lines drawn along the linear region of the FAM curves were compared as a measure of the steepness of the curve. However, we used the linear phase of the sigmoidal curve instead of its logarithm [22]. The suppression was proportional to the dye concentration. The higher the S82 increment was in the reaction the more flat the amplification curves were which was reflected in decreasing k values. The FAM suppression was the most remarkable after comparing the two extreme dye concentration points (k = 1077.9 vs. 337.7 for the FMDV and k = 1086.4 vs. 273.02 fort the IAV assay). Nevertheless, the FAM curves retained the sigmoidal shape even at the highest S82 concentration tested.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0151204.g001: 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.
Mentions: The evaluation of the S82 gradient showed that increasing concentration of the dye had a remarkable suppressive effect on the FAM fluorescence curve (Fig 1A; Table A and Fig BA in S2 File). To quantify the observed changes, the slope values, k, of the lines drawn along the linear region of the FAM curves were compared as a measure of the steepness of the curve. However, we used the linear phase of the sigmoidal curve instead of its logarithm [22]. The suppression was proportional to the dye concentration. The higher the S82 increment was in the reaction the more flat the amplification curves were which was reflected in decreasing k values. The FAM suppression was the most remarkable after comparing the two extreme dye concentration points (k = 1077.9 vs. 337.7 for the FMDV and k = 1086.4 vs. 273.02 fort the IAV assay). Nevertheless, the FAM curves retained the sigmoidal shape even at the highest S82 concentration tested.

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
Related in: MedlinePlus