Nagy Alexander, Černíková Lenka, Vitásková Eliška, Křivda Vlastimil, Dán Ádám, Dirbáková Zuzana, Jiřincová Helena, Procházka Bohumír, Sedlák Kamil, Havlíčková Martina
Laboratory of Molecular Methods, State Veterinary Institute Prague, Prague, Czech Republic.
National Reference Laboratory for Influenza, National Institute of Public Health, Prague, Czech Republic.
PLoS One. 2016 Mar 31;11(3):e0151204. doi: 10.1371/journal.pone.0151204. eCollection 2016.
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.
在本研究中，我们优化并评估了一种qPCR系统，该系统在单个反应中整合了6-羧基荧光素（6-FAM）标记的TaqMan探针，并使用SYTO 82（S82）DNA结合染料进行熔解分析。我们研究了S82对各种TaqMan和熔解分析参数的影响，并确定了其最佳浓度。下一步，使用包括现场样本在内的各种DNA和RNA模板，在36种不同的TaqMan检测中对该方法进行了评估，共进行了729对反应。此外，将感兴趣的熔解曲线与电泳图谱进行了关联。我们证明了S82与FAM-TaqMan系统完全兼容。此外，通过实际例子说明了这种方法在常规诊断TaqMan qPCR中的优势。这些优势包括解决平坦或其他非典型扩增曲线的问题，甚至解决由于探针结合失败导致的假阴性问题。我们的数据清楚地表明，将TaqMan qPCR和熔解分析整合到单个检测中，提供了额外的对照选项，以及进行更复杂分析、从反应中获取更多数据并以更高置信度获得分析结果的机会。
