Method for the efficiency-corrected real-time quantification of nucleic acids

Abstract

The present invention concerns a method for the quantification of a target nucleic acid in a sample comprising the following steps: (i) determination of the amplification efficiency of the target nucleic acid under defined amplification conditions, (ii) amplification of the target nucleic acid contained in the sample under the same defined reaction conditions, (iii) measuring the amplification in real-time, (iv) quantification of the original amount of target nucleic acid in the sample by correction of the original amount derived from step (iii) with the aid of the determined amplification efficiency. The efficiency correction of PCR reactions according to the invention for the quantification of nucleic acids can be used for absolute quantification with the aid of an external or internal standard as well as for relative quantification compared to the expression of housekeeping genes.

What is claimed is:  
     
         1 . A method for the quantification of a target nucleic acid in a sample comprising the steps of: 
 a) determination of the amplification efficiency of the target nucleic acid under defined amplification conditions;    b) amplification of the target nucleic acid contained in the sample under the same defined reaction conditions;    c) measuring the amplification in real-time; and    d) quantification of the original amount of target nucleic acid in the sample by correction of the original amount derived from step c) with the aid of the determined amplification efficiency.    
     
     
         2 . The method of  claim 1 , wherein the efficiency of the amplification is determined by: 
 a) preparing a dilution series of the target nucleic acid;    b) amplifying the target nucleic acid under defined reaction conditions as claimed in  claim 1 , the amplification of the nucleic acid being measured in real time;    c) determining a defined threshold value;    d) determining the cycle number at which the signal threshold value is exceeded for each dilution;    e) determining a logarithmic linear function of the copy number of target nucleic acid used for the amplification as a function of the cycle number at which the signal threshold value is exceeded; and    f) calculating the amplification efficiency E according to      E=G   −a      wherein  a  is determined as the first derivative of the function determined in step e) and G is the base number of the logarithm.    
     
     
         3 . The method of  claim 1 , wherein the efficiency of the amplification is determined by: 
 a) preparing a dilution series of the target nucleic acid;    b) amplifying the target nucleic acid under defined reaction conditions as claimed in  claim 1 , the amplification of the nucleic acid being measured in real time;    c) determining a defined signal threshold value;    d) determining the cycle number at which the signal threshold value is exceeded for each dilution;    e) determining a linear function of the cycle number determined in step d) as a function of a logarithm of the copy number of target nucleic acid used for the amplification; and    f) calculating the amplification efficiency E according to      E=G   −1/a      wherein  a  is determined as the first derivative of the function determined in step e) and G is the base number of the logarithm.    
     
     
         4 . The method of  claim 1  wherein the efficiency of the amplification is determined by: 
 a) preparing a dilution series of the target nucleic acid;  
 b) amplifying the target nucleic acid under defined reaction conditions as claimed in  claim 1 , the amplification of the nucleic acid being measured in real time;  
 c) determining a defined signal threshold value;  
 d) determining the cycle number at which the signal threshold value is exceeded for each dilution; and  
 e) determining the amplification efficiency as a function of the amount of target nucleic acid.  
 
     
     
         5 . A method for the quantification of a target nucleic acid in a sample relative to a reference nucleic acid comprising the steps of: 
 a) determination of the amplification efficiencies of the target nucleic acid and of the reference nucleic acid under defined amplification conditions;    b) amplification of the target nucleic acid contained in the sample and of the reference nucleic acid contained in the sample under the same defined reaction conditions;    c) measuring the amplification of the target nucleic acid and of the reference nucleic acid in real time; and    d) calculation of the original ratio of target nucleic acid and reference nucleic acid in the sample by correction of the ratio derived from step c) with the aid of the amplification efficiencies determined in step a).    
     
     
         6 . The method of  claim 5 , wherein steps b)-d) are additionally carried out using a calibrator sample and subsequently the ratio of the quotients determined for the sample and for the calibrator sample is determined as a measure for the original amount of target nucleic acid in the sample.  
     
     
         7 . A method for the quantification of a target nucleic acid in a sample relative to a reference nucleic acid comprising the steps of: 
 a) determination of the amplification efficiencies of the target nucleic acid and of the reference nucleic acid under defined amplification conditions;    b) amplification of the target nucleic acid contained in the sample as well as of the reference nucleic acid contained in the sample under the same defined amplification conditions;    c) measurement of the amplification of the target nucleic acid and of the reference nucleic acid in real time;    d) determining a defined signal threshold value;    e) determining the cycle numbers at which the signal threshold value is exceeded in each case during the amplification of target nucleic acid and reference nucleic acid; and    f) calculating the original ratio of target nucleic acid and of reference nucleic acid in the sample according to the following formula      N ( T ) 0   /N ( R ) 0   =E ( R ) n ( R )/ E ( T ) n ( T ), in which    N(T) 0 =the original amount of target DNA present in the sample    N(R) 0 =the original amount of reference DNA present in the sample    E(R)=the amplification efficiency of the reference nucleic acid    n(R)=the cycle number of the reference nucleic acid measured in step e)    E(T)=the amplification efficiency of the target nucleic acid    n(T)=the cycle number of the target nucleic acid measured in step e).    
     
     
         8 . The method of  claim 7 , wherein 
 steps b), c), e) and f) are additionally carried out using a calibrator sample and subsequently the ratio of the quotients determined for the sample and for the calibrator sample is determined as a measure for the original amount of target nucleic acid in the sample.    
     
     
         9 . The method of  claim 7 , wherein the real-time measurement of the amplification of target nucleic acid and reference nucleic acid in a sample is carried out in separate reaction vessels.  
     
     
         10 . The method of  claim 7 , wherein the real-time measurement of the amplification of target nucleic acid and reference nucleic acid in a sample is carried out in the same reaction vessel using differently labelled hybridization probes.  
     
     
         11 . A method for the quantification of a target nucleic acid in a sample comprising the steps of: 
 a) determination of the amplification efficiencies of the target nucleic acid and of an internal or external standard under defined amplification conditions;    b) amplification of the target nucleic acid contained in the sample and of the internal or external standard under the same defined reaction conditions;    c) measuring the amplification of the target nucleic acid and of the standard in real time; and    d) calculating the original copy number in the sample by correction of the copy number derived from step c) with the aid of the amplification efficiencies determined in step a).    
     
     
         12 . A method for the quantification of a target nucleic acid in a sample comprising the steps of: 
 a) determination of the amplification efficiencies of the target nucleic acid and of an internal or external standard under defined amplification conditions;    b) amplification of the target nucleic acid contained in the sample as well as of the internal or external standard under the same defined reaction conditions;    c) measurement of the amplification of the target nucleic acid and of the standard in real time;    d) determining a defined signal threshold value;    e) determining the cycle numbers at which the signal threshold value is exceeded during the amplification of target nucleic acid and standard; and    f) determining the original copy number N(T) 0  of target nucleic acid in the sample according to the formula      N ( T ) 0   =N ( S ) 0   *E ( S ) n(S)   /E ( T ) n(T) , in which    N(S) 0 =the original amount of standard used    E(S)=the amplification efficiency of the standard    n(S)=the cycle number of the standard measured in step e)    E(T)=the amplification efficiency of the target nucleic acid    n(T)=the cycle number of the target nucleic acid measured in step e).    
     
     
         13 . The method of  claim 12  using an internal standard, wherein real-time measurement of the amplification of the target nucleic acid and internal standard is carried out with differently labelled hybridization probes.  
     
     
         14 . The method of  claim 12 , wherein the amplified nucleic acids are detected with the aid of at least one fluorescent-labelled hybridization probe.  
     
     
         15 . The method of  claim 14 , wherein the amplified nucleic acids are detected with the aid of FRET hybridization probes, molecular beacons or TaqMan probes.  
     
     
         16 . The method of  claim 12 , wherein the amplified nucleic acids are detected with the aid of a DNA-binding dye, preferably with SybrGreen I.  
     
     
         17 . A kit containing agents to carry out the method of  claim 12.