Multiple digital PCR concentration measuring method and microdroplet type digital PCR system

Abstract

The invention discloses a multiple digital PCR concentration measuring method and a microdroplet type digital PCR system. The multiple digital PCR concentration measuring method is suitable for detecting concentrations of two or above target spots in a PCR reaction sample at the same time; the different target spots are marked by using different fluorophores; the multiple digital PCR concentrationmeasuring method comprises the steps of determining a filter bandwidth range corresponding to each fluorophore so as to determine a corresponding channel unit; detecting luminous intensity of each channel unit; obtaining a luminous contribution ratio of each fluorophore in the channel unit according to luminous spectrum of the fluorophore and the corresponding filter bandwidth range of the fluorophore; and obtaining the concentration of each fluorophore according to the luminous intensity of the channel unit and the luminous contribution ratio of each fluorophore in the channel unit. The invention provides a PCR concentration calculation method; and through algorithm correction, gray spots observed in the channels are effectively reduced, and fluorescence crosstalk between the channels caused by multiple target spots are corrected.

Description

technical field [0001] The invention relates to the technical field of PCR, in particular to a multiple digital PCR concentration measurement method and a droplet digital PCR system. Background technique [0002] The current digital PCR technology usually divides the sample into a large number of micro-reaction chambers with a volume of nanoliters in some way, and then performs PCR cycles on these micro-reaction chambers at the same time. When the PCR amplification enters the saturation period, the end-point observation method is adopted. The results of each reaction chamber are distinguished as negative or positive reactions. However, in practical applications, it is usually necessary to simultaneously detect two or more targets (amplification targets) in the sample, so it is necessary to use different fluorophores (which emit fluorescence in a specific band after being excited) to label different target. In order to distinguish the fluorophores of different targets, two ...

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Problems solved by technology

But in fact, the emission bands of fluorophores are difficult to avoid without overlapping, such as figure 1 As shown in the figure, the emission band of the HEX fluorophore is almost covered by the emission band of the FAM fluorophore, so no matter how to optimize the central wavelength and bandwidth of the detection channel, each channel will detect different fluorophores. This phenomenon fluorescence crosstalk between different channels

This kind of crosstalk will greatly interfere with the negative and positive judgment of the reaction chamber in different observation channels

In addition, the reactions of different targets in the same reaction chamber will form competition, which will affect the reaction efficiency of a single target, and then affect the end point observation of the fluorophore marking the target, resulting in a negative and positive judgment error

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