title: MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay.
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.
authors.affiliation: Laboratory of Molecular Methods, State Veterinary Institute Prague, Prague, Czech Republic.|National Reference Laboratory for Influenza, National Institute of Public Health, Prague, Czech Republic.
authors.forename: Alexander
authors.initials: A
authors.lastname: Nagy
authors.affiliation: Laboratory of Molecular Methods, State Veterinary Institute Prague, Prague, Czech Republic.
authors.forename: Lenka
authors.initials: L
authors.lastname: Černíková
authors.affiliation: Laboratory of Molecular Methods, State Veterinary Institute Prague, Prague, Czech Republic.
authors.forename: Eliška
authors.initials: E
authors.lastname: Vitásková
authors.affiliation: Laboratory of Molecular Methods, State Veterinary Institute Prague, Prague, Czech Republic.|Department of Virology and Serology, State Veterinary Institute Prague, Prague, Czech Republic.
authors.forename: Vlastimil
authors.initials: V
authors.lastname: Křivda
authors.affiliation: National Food Chain Safety Office, Veterinary Diagnostic Directorate, Molecular Biology Laboratory, Budapest, Hungary.
authors.forename: Ádám
authors.initials: Á
authors.lastname: Dán
authors.affiliation: Department of Virology, State Veterinary Institute Zvolen, Zvolen, Slovak Republic.
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Dirbáková
authors.affiliation: National Reference Laboratory for Influenza, National Institute of Public Health, Prague, Czech Republic.
authors.forename: Helena
authors.initials: H
authors.lastname: Jiřincová
authors.affiliation: Department of Informatics and Biostatistics, National Institute of Public Health, Prague, Czech Republic.
authors.forename: Bohumír
authors.initials: B
authors.lastname: Procházka
authors.affiliation: Department of Virology and Serology, State Veterinary Institute Prague, Prague, Czech Republic.
authors.forename: Kamil
authors.initials: K
authors.lastname: Sedlák
authors.affiliation: National Reference Laboratory for Influenza, National Institute of Public Health, Prague, Czech Republic.
authors.forename: Martina
authors.initials: M
authors.lastname: Havlíčková
mesh_terms: D015342:DNA Probes / Q000737:chemistry / Q000378:metabolism; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics; D009980:Influenza A virus / Q000235:genetics; D009696:Nucleic Acids / Q000378:metabolism*; D044367:Phase Transition; D012367:RNA, Viral / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase / Q000378:metabolism
chemical_list: D015342:DNA Probes; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009696:Nucleic Acids; D012367:RNA, Viral; C024098:6-carboxyfluorescein; D019914:Taq Polymerase