Second-derivative method for quantification cycle (Cq) determination.

Top panel: Fluorescence trace for a BEARmix reaction containing 250 N gene RNA molecules, showing a slow upward drift in baseline fluorescence prior to the onset of detectable amplification. Middle panel: Derivative of fluorescence intensity with respect to cycle number, calculated over a sliding window of ±3 cycles. Bottom panel: Second derivative of the fluorescence intensity, i.e., derivative of the curve in the middle panel. The second derivative is zero during the initial phase of linear baseline drift and peaked near the onset of detectable amplification. Red vertical line: Cq value, determined as the center of a parabolic fit to the peak of the second derivative curve.

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