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

• Published in: PloS one Vol. 11; no. 3; p. e0151204
• Main Authors: Nagy, Alexander, Černíková, Lenka, Vitásková, Eliška, Křivda, Vlastimil, Dán, Ádám, Dirbáková, Zuzana, Jiřincová, Helena, Procházka, Bohumír, Sedlák, Kamil, Havlíčková, Martina
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 31-03-2016; Public Library of Science (PLoS)
• Subjects: Assaying; Binding; Biology and life sciences; Confidence intervals; Data processing; Deoxyribonucleic acid; Diagnostic systems; DNA; DNA probes; DNA Probes - chemistry; DNA Probes - metabolism; Dyes; Fluoresceins - chemistry; Fluorescent Dyes - chemistry; Foot-and-Mouth Disease Virus - genetics; Genetic analysis; Influenza; Influenza A virus - genetics; Laboratories; Medicine and health sciences; Melting; Molecular biology; Mutation; Nucleic Acids - metabolism; Optimization; Phase Transition; Physical Sciences; Polymerase chain reaction; Public health; Real-Time Polymerase Chain Reaction - methods; Research and Analysis Methods; Ribonucleic acid; RNA; RNA, Viral - metabolism; Taq Polymerase - metabolism; Virology; Viruses; Czech Republic
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0151204

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.