Compensation for spectral interference in multiplex nucleic acid amplification

摘要

Method comprising: a calibration process comprising: - performing at least one singleplex nucleic acid amplification of a nucleic acid sample using a detection probe, in which the nucleic acid amplification occurs during a plurality of interrogation periods ; - from the nucleic acid amplification, acquire amplification data indicating an amount of nucleic acid present for each of the plurality of interrogation periods; and - based on the amplification data, determine an interference correction value associated with a spectral neighbor closest to the detection probe to reduce the spectral interference of the nearest spectral neighbor; the method further comprising a nucleic acid amplification analysis that includes applying the interference correction value obtained from the calibration process to the amplification data collected from the multiplex nucleic acid amplification of nucleic acid samples, in which the Interference correction value determined is the proportion of a signal detected for the nearest spectral neighbor with respect to the signal detected for the amplification data of the detection probe, in which the nearest spectral neighbor is determined based on a distance from a fluorescence peak of the target dye of the detection probe to the fluorescence peak of a neighboring target dye, wherein the fluorescence peak of the target dye of the detection probe and the fluorescence peak of the neighboring target dye nearest spectral are separated by a distance less than a dis The threshold value, and in which applying the interference correction value to the amplification data comprises: - subtracting a background signal from the signal detected for the amplification data to obtain a signal corrected by the background; and - subtracting, from the signal corrected by the background, a product of the interference correction value and the signal detected by the nearest spectral neighbor.