PCR measuring method and measurement device

A method for determining whether or not each of a plurality of target DNAs is contained in each of a plurality of droplets in oil, each droplet containing a plurality of fluorescent labeled probes, each fluorescent labeled probe comprising a fluorescent dye and a quencher conjugated at or in a vicinity of respective ends thereof being hybridized to a corresponding target DNA of the plurality of target DNAs between the fluorescent dye and the quencher, and being configured to form a stem-loop structure by sequences near both ends, the sequences being complementary to each other, the plurality of fluorescent labeled probes comprises at least a first fluorescent labeled probe and a second fluorescent labeled probe, wherein the first and second fluorescent labeled probes each comprise a first fluorescent dye associated with a first fluorescence color, wherein the first fluorescent labeled probe is configured for hybridization to a first target DNA and the second fluorescent labeled probe is configured for hybridization to a second target DNA of the plurality of target DNAs that is different from the first target DNA, the method comprising:

• a first step of performing a nucleic acid amplification reaction in the plurality of droplets;
• a second step of, after completion of the nucleic acid amplification reaction, measuring fluorescence intensity associated with the plurality of fluorescent labeled probes in each droplet of the plurality of droplets at a plurality of increasing temperatures, wherein the fluorescence intensity associated with the plurality of fluorescent labeled probes comprises a fluorescence intensity associated with the first fluorescence color;
• a third step of calculating, for each droplet in the plurality of droplets with a measured fluorescence intensity above a threshold fluorescence intensity, at least one melting temperature (Tm) associated with at least one fluorescent labeled probe of the plurality of fluorescent labeled probes in the droplet by identifying at least one corresponding inflection point of the measured fluorescence intensity of the at least one fluorescent labeled probe;
• a fourth step of determining,
• that the first target DNA is not contained in droplets in a first set of droplets in the plurality of droplets based on (1) the measured fluorescence intensity being below the threshold fluorescence intensity or (2) the measured fluorescence intensity being above the threshold fluorescence intensity and a first calculated Tm of the at least one calculated Tm being out of a first predetermined range of Tm around a first reference Tm calculated for the first fluorescent labeled probe of the at least one fluorescent labeled probe and the first target DNA of the plurality of target DNAs in bulk, and
• that the second target DNA is not contained in droplets in a second set of droplets in the plurality of droplets based on (1) the measured fluorescence intensity being below the threshold fluorescence intensity or (2) the measured fluorescence intensity being above the threshold fluorescence intensity and a second calculated Tm of the at least one calculated Tm being out of a second predetermined range of Tm around a second reference Tm calculated for the second fluorescent labeled probe of the at least one fluorescent labeled probe and the second target DNA of the plurality of target DNAs in bulk, wherein the first reference Tm is different from the second reference Tm;
• a fifth step of counting:
• a first number of droplets in a third set of droplets in the plurality of droplets, wherein the third set of droplets are determined to contain the first target DNA based on (1) the measured fluorescence intensity being equal to, or larger than, the threshold fluorescence intensity and (2) the first calculated Tm being in the first predetermined range of Tm around the first reference Tm, and
• a second number of droplets in a fourth set of droplets in the plurality of droplets, wherein the fourth set of droplets are determined to contain the second target DNA based on (1) the measured fluorescence intensity being equal to, or larger than, the threshold fluorescence intensity and (2) the second calculated Tm being in the second predetermined range of Tm around the second reference Tm;
• and
• a sixth step of displaying, on a monitor, a set of values related to (1) a third number of droplets in an intersection of the first set of droplets and the second set of droplets that do not contain at least one of the first target DNA and the second target DNA, (2) the first number of droplets in the third set of droplets that contain the first target DNA, and (3) the second number of droplets in the fourth set of droplets that contain the second target DNA.