Figure 1.
Relationship between cycle threshold and melting temperature.
The relationship between cycle threshold (CT) and melting temperature (Tm) demonstrated by ten-fold serial dilutions of wildtype (H) (A/Victoria/2048/2009) and mutant (Y) (A/Osaka/180/2009) virus samples. A. The CT increases as the RNA template concentration decreases. The no template control (NTC) demonstrates late amplification after cycle 30, indicating the presence of primer-dimer. B. The Tm on the high-resolution melting normalized fluorescence graph decreases as the RNA template concentration decreases. The NTC melts at the lowest temperature. As the amplified product contains increasing proportions of the primer-dimer, the overall Tm is pulled towards that of the primer-dimer.
Figure 2.
Plot of the melting temperature (Tm) against the cycle threshold (CT).
The blue, red, and green lines are the quadratic fitted lines with 95% confidence intervals for dilutions (blue, red and green circles) of the controls of wildtype (H@275) (A/Victoria/2048/2009), mutant (Y@275) (A/Osaka/180/2009), and 30∶70 mix of mutant∶wildtype respectively. The 10∶90 mutant∶wildtype consistently plotted below the wildtype fitted curve, but at the lower border of the 95% confidence interval. All 56 clinical samples that had a CT≤30 correlated with the wildtype virus (28 confirmed by pyrosequencing). Blinded WHO samples (four wildtype and four mutant containing) and dilutions of mutant virus (A/Denmark/528/2009 and A/Perth/262/2009) fit within the expected curves. One blinded sample (A/Perth/268/2009) plotted just below the 30∶70 curve and pyrosequencing determined this to be a 34∶66 mix of mutant∶wildtype. Raw data is provided in Results S1.