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Rolf Knobel Roche Diagnostics International, Switzerland |
Abstract
For the automatic analysis of kinetic PCR data the regression of a mathematical curve function to the measurement signals appears attractive. The derived curve function is robust against signal imprecision and curve characteristics like a Cq value can be calculated easily. However, simple mathematical models like the logistic or Richard function fail to globally fit real kinetic PCR data well in many cases. Here, a novel mathematical function for kinetic PCR curve regression is introduced. It shows appropriate asymptotic behavior and can deal with different curvatures in the increase and the saturation phase of the reaction. Additionally, the application of this function for automatic PCR result calculation is shown: The calculation of the asymptotic signal growth size is useful for the correction of the spectral crosstalk of cleaved TaqMan products. A drifting baseline can be derived from the mathematical model curve as well in order to standardize the parallel curve display of many curves measured in different wells. For Cq calculation the intersection of a threshold line with the model function is insensitive to signal imprecision. Additionally, an automated qualitative judgment based on several curve characteristics like growth and maximal slope is possible. With the help of pre-set algorithm cutoffs the automatic calculation of the results can be adjusted for different applications like intercalation or multicolor TaqMan on each detection channel of an instrument. For the LightCycler® 96 thousands of real data sets have been used for training the algorithm parameters accordingly. So, the result calculation of kinetic PCR reactions can be performed fully automatic in a reliable way. LightCycler® is a trademark of Roche.
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