pixl Real-Time PCR Platform
Instructions for Use Manual
Page 24 of 37
pixl Real-Time PCR Instructions for Use Manual
Rev. 01-28JUL2021
Appendix A: PCR data processing – Positive/negative
determination and Ct calculation.
For each reaction well and each channel, a series of cycle-by-cycle fluorescence read out
data is obtained. The fluorescence read out from the reaction is processed to determine
whether the result is positive or negative. This process involves several steps:
1.
Alignment of the initial phase of the data. The algorithm estimates the average
signal magnitude of the data at the initial cycles and estimate the mean. For
subsequent processing, the algorithm will subtract the mean value to align the initial
phase of the data along the x axis of the coordinate systems.
2.
Estimate the standard deviation of the data in the initial phase of the data points.
3.
Determine and refine Ct value
The algorithm will then compare the magnitude of the data to the Ct threshold
value. The Ct threshold is determined with a multiple of the base line standard
deviation. If the magnitude of the data consistently exceeds the threshold, the result
is considered positive, the cycle number location of Ct threshold crossing is the Ct
value of the data.
Figure A1
. Ct value calculation using Ct threshold crossing.