Progress and melting curves of PCR products used for the detection of F. culmorum and F. graminearum. PCR and melting curve analysis was performed in a real-time thermocycler with primer pairs specific for F. culmorum and F. graminearum and SYBR Green I fluorescence detection as described in the Methods. The SYBR Green I was diluted to 0.4× the recommended concentration. Genomic DNA from F. graminearum (continuous curves with squares), F. culmorum (dotted curves with diamonds), a mixture containing DNA from both species (dashed curves with triangles) and no DNA (no-template control, "NTC ", gray curve without symbols) were used as templates. A: Progress curves, showing the fluorescence signal during the annealing phase in each reaction cycle. B: Melting curves recorded on PCR products after 35 cycles. The rise of fluorescence in the no-template control produces a melting point maximum at about 80°C, far lower than where Fusarium target are detected. Because melting curves presented in Figure 1B were recorded with low template amounts, the unspecific products are also visible, but they do not prevent the detection of Fusarium DNA in amounts equal to or higher than the limit of detection.
Brandfass and Karlovsky BMC Microbiology 2006 6:4 doi:10.1186/1471-2180-6-4