Patent ID: 8024132

Claim:
A method for the quantification of a target nucleic acid in a test sample relative to a reference nucleic acid by using a calibrator sample, wherein the calibrator sample comprises the target nucleic acid and the reference nucleic acid and the test sample comprises the target nucleic acid and the reference nucleic acid, comprising: a) determining amplification efficiencies of the target nucleic acid and of the reference nucleic acid under defined amplification conditions, b) amplifying the target nucleic acid contained in the test sample and the reference nucleic acid contained in the test sample under the same defined reaction conditions, c) measuring the amplification of the target nucleic acid from the test sample and of the reference nucleic acid from the test sample in real time to determine amplified amounts of the target nucleic acid and the reference nucleic acid from the test sample, d) amplifying the target nucleic acid contained in a calibrator sample and the reference nucleic acid contained in the calibrator sample under the same defined reaction conditions, e) measuring the amplification of the target nucleic acid from the calibrator sample and of the reference nucleic acid from the calibrator sample in real time to determine amplified amounts of the target nucleic acid and the reference nucleic acid from the calibrator sample, and f) combining the amplified amounts determined for the sample and for the calibrator sample in steps c) and e) with the aid of the amplification efficiencies determined in step a), wherein the combining is based upon the formula N ⁡ ( T ) 0 ⁢ A N ⁡ ( R ) 0 ⁢ A / N ⁡ ( T ) 0 ⁢ K N ⁡ ( R ) 0 ⁢ K = E ⁡ ( R ) nA ⁡ ( R ) - nK ⁡ ( R ) * E ⁡ ( T ) nK ⁡ ( T ) - nA ⁡ ( T ) , wherein N(T) 0i =the original amount of target DNA present N(R) 0i =the original amount of reference DNA present E(R)=the amplification efficiency of the reference nucleic acid ni(R)=the cycle number of the reference nucleic acid E(T)=the amplification efficiency of the target nucleic acid ni(T)=the cycle number of the target nucleic acid and i=A are values of the test sample and i=K are values of the calibrator sample, g) determining a measure for the original amount of target nucleic acid in the sample using the formula in step f).