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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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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.
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pone.0151204.g004: 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.

Mentions: The results of all seven TaqMan assays are summarised in Table D and Fig F in S2 File. Overall, no significant differences were observed between the standard and test subsets, with both yielding highly similar Cq values (Fig 4) and low standard deviation values suggesting low intra-assay or intra-subset variation.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0151204.g004: 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.
Mentions: The results of all seven TaqMan assays are summarised in Table D and Fig F in S2 File. Overall, no significant differences were observed between the standard and test subsets, with both yielding highly similar Cq values (Fig 4) and low standard deviation values suggesting low intra-assay or intra-subset variation.

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