Systems and methods are described for quantifying a target nucleic acid. A sample comprising a target nucleic acid is segregated into a first plurality of the reaction volumes containing at least one target nucleic acid molecule and a second plurality of the reaction volumes containing no target nucleic acid molecules. The reaction volumes are subjected to an amplification assay, wherein the amplification assay is configured to amplify the target nucleic acid. An indicator of amplification is detected or measured in at least some of the plurality of reaction volumes. The target nucleic acid is quantified based on the detection or measurement. After discontinuing the amplification assay, the plurality of reaction volumes may be heated and changes in the indicators of amplification of two or more of the at least some of the reaction volumes may be detected or measured.

Systems and methods are described for quantifying a target nucleic acid. A sample comprising a target nucleic acid is segregated into a first plurality of the reaction volumes containing at least one target nucleic acid molecule and a second plurality of the reaction volumes containing no target nucleic acid molecules. The reaction volumes are subjected to an amplification assay, wherein the amplification assay is configured to amplify the target nucleic acid. An indicator of amplification is detected or measured in at least some of the plurality of reaction volumes. The target nucleic acid is quantified based on the detection or measurement. After discontinuing the amplification assay, the plurality of reaction volumes may be heated and changes in the indicators of amplification of two or more of the at least some of the reaction volumes may be detected or measured.

The invention(s) cover systems and methods for target detection in a multiplexed and rapid manner. Embodiments of the system can include: a base substrate; and an array of sample processing regions defined at a broad surface of the base substrate, wherein each of the array of sample processing regions includes: a set of microwell subarrays arranged in a gradient by volumetric capacity between an upstream end and a downstream end of each respective sample processing region, and a boundary separating each respective sample processing region from adjacent sample processing regions. The system can support methods, with example implementation by an automated platform, for returning preliminary results from a subset of microwells of the samples processing regions, as well as results pertaining to specific and non-specific amplification, for multiple targets of a sample.

The invention(s) cover systems and methods for target detection in a multiplexed and rapid manner. Embodiments of the system can include: a base substrate; and an array of sample processing regions defined at a broad surface of the base substrate, wherein each of the array of sample processing regions includes: a set of microwell subarrays arranged in a gradient by volumetric capacity between an upstream end and a downstream end of each respective sample processing region, and a boundary separating each respective sample processing region from adjacent sample processing regions. The system can support methods, with example implementation by an automated platform, for returning preliminary results from a subset of microwells of the samples processing regions, as well as results pertaining to specific and non-specific amplification, for multiple targets of a sample.

A method for template-directed sequencing-by-synthesis of an array of target polynucleotide can include: (a) providing an array of target polynucleotides in a fluidic vessel; (b) contacting the array of polynucleotides with a solution comprising (i) polymerization complex and (ii) reversibly terminating and differently labeled A, C, G, and T/U nucleotides; (c) incorporating one of the differently labeled nucleotides, using the polymerization complex, into a chain complementary to at least one of the array of polynucleotides; (d) binding imaging tags to the differently labeled nucleotides of step (c); (e) imaging and storing the identity and position of the imaging tags of step (d); (f) reversing termination (b)-(e); (g) repeating steps (b)-(e) and assembling a sequence for each of the array of target polynucleotides from the stored identity and position of the imaging tags, optionally as a homogeneous or one pot reaction. Additional methods of sequencing target polynucleotides are described herein.

A method for template-directed sequencing-by-synthesis of an array of target polynucleotide can include: (a) providing an array of target polynucleotides in a fluidic vessel; (b) contacting the array of polynucleotides with a solution comprising (i) polymerization complex and (ii) reversibly terminating and differently labeled A, C, G, and T/U nucleotides; (c) incorporating one of the differently labeled nucleotides, using the polymerization complex, into a chain complementary to at least one of the array of polynucleotides; (d) binding imaging tags to the differently labeled nucleotides of step (c); (e) imaging and storing the identity and position of the imaging tags of step (d); (f) reversing termination (b)-(e); (g) repeating steps (b)-(e) and assembling a sequence for each of the array of target polynucleotides from the stored identity and position of the imaging tags, optionally as a homogeneous or one pot reaction. Additional methods of sequencing target polynucleotides are described herein.

Disclosed herein are methods and compositions for determining the copy number of a first target nucleic acid as compared to the copy number of a second target nucleic acid in a single well with a single detection label. For example, disclosed herein are methods and compositions for determining the copy number of a first target nucleic acid as compared to the copy number of a second target nucleic acid by a monochrome multiplex quantitative PCR (MMQPCR) in a single well with a single detection label.

Disclosed herein are methods and compositions for determining the copy number of a first target nucleic acid as compared to the copy number of a second target nucleic acid in a single well with a single detection label. For example, disclosed herein are methods and compositions for determining the copy number of a first target nucleic acid as compared to the copy number of a second target nucleic acid by a monochrome multiplex quantitative PCR (MMQPCR) in a single well with a single detection label.

Disclosed herein are primers and probes related to the detection of SARS-CoV-2 via nucleic acid amplification testing (NAAT), for example to amplify and determine the presence of SARS-CoV-2 in test samples and/or to diagnose Covid-19. Specifically, the present disclosure describes primers and probes that bind to the N gene, ORF1ab, or E gene of SARS-CoV-2 coronavirus for detection via loop mediated isothermal amplification (LAMP) and molecular beacon hybridization.

Disclosed herein are primers and probes related to the detection of SARS-CoV-2 via nucleic acid amplification testing (NAAT), for example to amplify and determine the presence of SARS-CoV-2 in test samples and/or to diagnose Covid-19. Specifically, the present disclosure describes primers and probes that bind to the N gene, ORF1ab, or E gene of SARS-CoV-2 coronavirus for detection via loop mediated isothermal amplification (LAMP) and molecular beacon hybridization.