MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay
Jazyk angličtina Země Spojené státy americké Médium electronic-ecollection
Typ dokumentu časopisecké články, práce podpořená grantem
PubMed
27031831
PubMed Central
PMC4816343
DOI
10.1371/journal.pone.0151204
PII: PONE-D-15-44447
Knihovny.cz E-zdroje
- MeSH
- DNA sondy chemie metabolismus MeSH
- fluoresceiny chemie MeSH
- fluorescenční barviva chemie MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- nukleové kyseliny metabolismus MeSH
- RNA virová metabolismus MeSH
- Taq-polymerasa metabolismus MeSH
- virus chřipky A genetika MeSH
- virus slintavky a kulhavky genetika MeSH
- změna skupenství MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 6-carboxyfluorescein MeSH Prohlížeč
- DNA sondy MeSH
- fluoresceiny MeSH
- fluorescenční barviva MeSH
- nukleové kyseliny MeSH
- RNA virová MeSH
- Taq-polymerasa MeSH
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 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
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