Method for detecting nucleic acid amplification product in real time
There is provided an apparatus for detecting a nucleic acid amplification product in real time, which is capable of effectively excluding or reducing apparatus error factors without using a second fluorescence signal used for correction. A plurality of wells 7 A are given with temperature cycles and fluorescence strength from a nucleic acid amplification product is detected in real time in each well 7 A. A fluorescence measurement value [DNA]raw obtained from the well 7 A and a fluorescence measurement value [DNA]bg obtained from a connection wall near the well 7 A are detected, and the fluorescence measurement value [DNA]bg is subtracted from the fluorescence measurement value [DNA]raw to determine fluorescence strength [DNA]real of the well 7 A.
1. A method for detecting fluorescence strength from a nucleic acid amplification product in real time in each of a plurality of reaction regions in a reaction vessel undergoing temperature cycling, comprising the steps of:
subjecting a reaction vessel having a plurality of reaction regions oriented in a rectangular array, to temperature cycling;
after a temperature cycle, detecting, using an optical system fluorescence measurement, separately for each of the reaction regions in the reaction vessel:
a value [DNA]raw from the reaction region; and
a fluorescence measurement value [DNA]bg, obtained from four separate regions, each of which is outside the reaction region and in a connection wall portion adjacent to the reaction region; and
subtracting the fluorescence measurement value [DNA]bg from the fluorescence measurement value [DNA]raw every time the fluorescence measurement value [DNA]raw is detected for each of the reaction regions separately to determine fluorescence strength [DNA]real of each of the reaction regions.
2. The method according to claim 1 ,
wherein the detecting and subtracting steps are performed after each temperature cycle.
3. The method according to claim 1 ,
wherein, in the detecting step, for each reaction region, the fluorescence measurement value [DNA]bg for the reaction region is a simple average value or a weighted average value of the four fluorescence measurement values from the four separate regions for that reaction region.