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.

Department of Informatics and Biostatistics National Institute of Public Health Prague Czech Republic

Department of Virology and Serology State Veterinary Institute Prague Prague Czech Republic

Department of Virology State Veterinary Institute Zvolen Zvolen Slovak Republic

Laboratory of Molecular Methods State Veterinary Institute Prague Prague Czech Republic

National Food Chain Safety Office Veterinary Diagnostic Directorate Molecular Biology Laboratory Budapest Hungary

National Reference Laboratory for Influenza National Institute of Public Health Prague Czech Republic

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PLoS One. 2018 Apr 23;13(4):e0196444. doi: 10.1371/journal.pone.0196444. PubMed

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