Apparatus for analysis of a nucleic acid amplification reaction

Abstract

An apparatus for determining a threshold value (e.g., a threshold cycle number or a time value) in a nucleic acid amplification reaction comprises a detection mechanism for measuring, at a plurality of different times during the amplification reaction, at least one signal whose intensity is related to the quantity of a nucleic acid sequence being amplified in the reaction. A controller in communication with the detection mechanism is programmed to store signal values defining a growth curve for the nucleic acid sequence, determine a derivative of the growth curve, and calculate a cycle number or time value associated with a characteristic of the derivative.

1 . An apparatus for determining a threshold time value in a nucleic acid amplification reaction, the apparatus comprising: a) a detection mechanism for measuring, at a plurality of different times during the amplification reaction, at least one signal whose intensity is related to the quantity of a nucleic acid sequence being amplified in the reaction; and b) a controller in communication with the detection mechanism, wherein the controller is programmed to perform the steps of: i) deriving a growth curve from the measurements of the signal; ii) calculating a derivative of the growth curve; iii) identifying a characteristic of the derivative; and iv) determining a time value associated with the characteristic of the derivative.  
   
   
       2 . The apparatus of  claim 1 , wherein the controller is programmed to calculate a second derivative of the growth curve, and wherein the characteristic comprises a positive peak of the second derivative.  
   
   
       3 . The apparatus of  claim 1 , wherein the controller is programmed to calculate a second derivative of the growth curve, and wherein the characteristic comprises a negative peak of the second derivative.  
   
   
       4 . The apparatus of  claim 1 , wherein the controller is programmed to calculate a second derivative of the growth curve, and wherein the characteristic comprises a zero crossing of the second derivative.  
   
   
       5 . The apparatus of  claim 1 , wherein the controller is programmed to calculate a first derivative of the growth curve, and wherein the characteristic comprises a positive peak of the first derivative.  
   
   
       6 . The apparatus of  claim 1 , wherein the controller is programmed to calculate second derivative values of the growth curve at a plurality of different measurement times in the reaction to yield a plurality of second derivative data points, the characteristic comprises a positive peak of the second derivative, and the controller is further programmed to determine the time value associated with the positive peak by: i) fitting a second order curve to the second derivative data points; and ii) calculating the time value as the location of a peak of the second order curve.