Apparatus for detecting nucleic acid amplification product in real time

Abstract

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 . An apparatus for detecting fluorescence strength from a nucleic acid amplification product in each of a plurality of reaction regions given with temperature cycles in real time,
 wherein a fluorescence measurement value [DNA]raw obtained from the reaction region and a fluorescence measurement value [DNA]bg obtained from regions other than the reaction region adjacent to the reaction region 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 reaction region.   
   
   
       2 . The apparatus according to  claim 1 ,
 wherein the fluorescence measurement value [DNA]bg is a simple average value of fluorescence measurement values obtained from the regions other than the plurality of reaction regions adjacent to the reaction region, or an average value of fluorescence measurement values after the fluorescence measurement values are weighted.   
   
   
       3 . The apparatus according to  claim 1  or  2 ,
 wherein the fluorescence measurement value [DNA]bg is detected every detection of the fluorescence measurement value [DNA]raw, and the fluorescence measurement value [DNA]bg is subtracted from the fluorescence measurement value [DNA]raw to determine the fluorescence strength [DNA]real.   
   
   
       4 . An apparatus for detecting fluorescence strength from a nucleic acid amplification product in each of a plurality of reaction regions given with temperature cycles in real time,
 wherein fluorescence strengths [DNA]n obtained from the reaction regions every temperature cycles n are normalized using the maximum value of the fluorescence strengths [DNA]n or related value [DNA]max, and a threshold cycle number Ct is calculated by setting a threshold Th in an exponential amplification region of an amplification curve drawn using a normalized fluorescence strength [DNA]nN.   
   
   
       5 . The apparatus according to  claim 4 ,
 wherein the fluorescence strength [DNA]nN is calculated by dividing the fluorescence strength [DNA]n for each reaction region by a value obtained by adding a value common to the reaction regions to be compared to the maximum value or related value [DNA]max.   
   
   
       6 . The apparatus according to  claim 4  or  5 ,
 wherein the amplification curve is drawn after fluorescence strength [DNA]base before the exponential amplification region is subtracted from the fluorescence strength [DNA]n for each reaction region and the maximum value or related value [DNA]max.