Methods for quantitative amplification

Abstract

Methods, sample vessels, and instruments are provided for quantitative and semi-quantitative amplification.

What is claimed is: 
     
         1 . A method for performing quantitative nucleic acid amplification on nucleic acid molecules in a sample, comprising
 a) lysing said sample;   b) extracting the nucleic acid molecules from said sample;   c) performing nucleic acid amplification of the nucleic acid molecules;   wherein at least one sample processing control is added to said sample prior to step a) or during step a) in a known amount;   characterized in that a nucleic acid sequence from the sample processing control serves as a quantification standard.   
     
     
         2 . The method according to  claim 1  wherein step c) comprises amplifying the nucleic acid molecule(s) contained in the sample with an amplification mixture, the amplification mixture comprising at least one target primer configured to amplify a target that may be present in the sample; and
 the amplification mixture further comprises at least one quantification standard primer set configured to amplify the quantification standard. 
 
     
     
         3 . The method according to  claim 2 , further comprising quantifying the target by comparing the signal obtained for the quantification standard and for the target with an imported standard curve. 
     
     
         4 . The method according to  claim 3 , wherein the imported standard curve has been generated using the least square regression line fit to:
   log 10 (concentration)=( Ct−b ′)/ a  
   wherein   Ct is a cycle threshold measured for each target,   a corresponds to the slope of the synthetic quantitative standard range and which represents the degree to which Ct changes with a single unit change in log 10  concentration, and   b′ corresponds to the calibrated intercept and representing the theoretical value of Ct when Log 10 (concentration) of SPC is zero when calibrated against the quantitative synthetic standard range.   
     
     
         5 . The method according to  claim 4 , further comprising applying a predetermined correction factor between the quantification standard and the target. 
     
     
         6 . The method according to  claim 5 , wherein the concentration of the target is determined according to the following equation:
   log 10 (concentration t )=log 10 (concentration s )+κ
   wherein the subscripts s and t respectively represent the sample processing control and the target and κ is a correction factor previously determined for said target.   
     
     
         7 . The method according to  6  wherein said quantitative amplification is quantitative PCR. 
     
     
         8 . The method according to  claim 7  wherein said amplification is multiplex PCR for the amplification of multiple targets. 
     
     
         9 . The method according to  claim 2  further comprising adding a second quantification standard prior to step c), and subsequent to step c) comparing amplification of the quantification standards. 
     
     
         10 . The method according to  claim 9  wherein a difference in amplification between the quantification standard and the second quantification standard is indicative of the efficiency of the step b).