METHODS FOR PERFORMING DIGITAL PCR
This invention releases to systems and methods for detecting the presence and quantity of a target nucleic acid in a sample using dPCR and PIP encapsulated monodisperse droplets.
1 . A method to detect a target nucleic acid, the method comprising:
preparing an aqueous solution comprising a target nucleic acid derived from a biological sample, PCR reagents, template particles, primers specific for the target nucleic acid, and fluorescent probes;
combining the aqueous solution with an oil in a vessel to create a mixture;
shearing the mixture to form a plurality of water-in-oil partitions, wherein each of the partitions includes a single target nucleic acid, PCR reagents, a template particle, primers specific for the target nucleic acid, and at least one fluorescent probe;
hybridizing the primers and fluorescent probes to the target nucleic acid;
amplifying the target nucleic acids in the partitions, thereby hydrolyzing the probes to release a fluorescent label;
identifying partitions with a fluorescent signal from the fluorescent label to detect the presence of the target nucleic acid in the sample.
2 . The method of claim 1 , wherein the water-in-oil partitions are formed simultaneously.
3 . The method of claim 1 , wherein the template particles template the formation of the droplets and segregate the microbial nucleic acid inside one of the droplets away from other nucleic acids present in the sample.
4 . The method of claim 1 , wherein the fluorescent signal is detected using a fluorometer.
5 . The method of claim 4 , wherein the fluorometer is a fluorescent cell counter.
6 . The method of claim 1 , further comprising quantifying the amount of target nucleic acid in the sample.
7 . The method of claim 6 , wherein quantifying the amount of target nucleic acid includes counting the number of droplets that produce a detectable signal and the number of droplets that do not produce a detectable signal.
8 . The method of claim 7 , wherein the target nucleic acid is loaded into the partitions at a limiting dilution.
9 . The method of claim 1 , wherein further comprising detecting the presence of two or more different target nucleic acids in a sample.
10 . The method of claim 9 , wherein shearing the mixture forms a plurality of partitions that each include one of the different target nucleic acids.
11 . The method of claim 10 , wherein the partitions include a plurality of hydrolysis probes, wherein each probe binds to a different target nucleic acid and includes a different fluorescent label.
12 . The method of claim 11 , wherein identifying the presence of the target nucleic acids in the sample includes imaging the partitions to detect the fluorescence emission of each different fluorescent label.
13 . The method of claim 12 , further comprising quantifying the amount of each different target nucleic acid in the sample.
14 . A method to detect microbial nucleic acid, the method comprising:
obtaining a sample comprising a microbial nucleic acid;
partitioning the sample to form a plurality of droplets simultaneously, wherein the microbial nucleic acid is segregated inside one of the droplets;
binding, inside the droplet, the microbial nucleic acid with a capture probe;
amplifying bound microbial nucleic acid to create an amplicon; and
detecting the amplicon to thereby detect the microbial nucleic acid.
15 . The method of claim 14 , wherein the microbial nucleic acid comprises 16s rDNA.
16 . The method of claim 14 , wherein amplifying the bound microbial nucleic acid is performed with PCR in the presence of a fluorophore and wherein said fluorophore is incorporated into the amplicon during amplification.
17 . The method of claim 16 , wherein the fluorophore comprises an intercalating dye.
18 . The method of claim 16 , wherein detecting the amplicon is achieved by sensing a fluorescent signal from the fluorophore, wherein the fluorescent signal is indicative of the amplicon.
19 . The method of claim 14 , further comprising:
combining template particles with the sample in a first fluid;
adding a second fluid that is immiscible with the first fluid to create a mixture; and
vortexing the mixture, thereby partitioning the sample to form the plurality of droplets.
20 . The method of claim 19 , wherein the template particles template the formation of the droplets and segregate the microbial nucleic acid inside one of the droplets away from other nucleic acids present in the sample.
21 - 50 . (canceled)