Patent ID: 7630837

Claim:
A method of determining mass fractions of first and second target nucleic acids present in a test sample comprising: obtaining an approximated standard melting signal, f i , for each of the first and second target nucleic acids, the approximated standard melting signal for the first target nucleic acid corresponding to a change in fluorescence of the first target nucleic acid over temperature and the approximated standard melting signal for the second target nucleic acid corresponding to a change in fluorescence of the second target nucleic acid over temperature, contacting the target nucleic acids in the test sample with a fluorescent nucleic acid indicator, the indicator being configured to provide a signal related to the quantity of indicator hybridized to the target nucleic acids, the indicator further configured to discriminate the target nucleic acids based on melting temperature, illuminating the test sample including the fluorescent nucleic acid indicator, cycling the illuminated test sample through an annealing temperature and a denaturing temperature that is higher than the annealing temperature, monitoring fluorescent change of the illuminated test sample while changing temperature of the illuminated test sample to obtain a fluorescence melting signal, f mix , of the test sample, the fluorescence melting signal, f mix , corresponding to a change in fluorescence of the test sample over temperature, and approximating the fluorescence melting signal, f mix , for the test sample over temperature, T, as a combination of the approximated standard melting signals, f i , of the first and second target nucleic acids, and using f i and f mix to determine the mass fractions, m i , of the first and second target nucleic acids in the test sample according to the formula ∫ T 0 T 1 ⁢  f mix - ∑ i ⁢ ⁢ m i ⁢ f i ( ⁢ T )  ⁢ ⅆ T .