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Publication Number
US-12503727-B2
Publication Date
2025-12-23
Expiration Date
Abstract
Digital assay system, including methods, apparatus, and compositions, for assay of one or more targets in a set of partitions containing a generic reporter of target amplification.
Core Innovation
The invention relates to performing a multiplexed digital assay by forming partitions, where only a first subset of the partitions contains a first target and only a second subset contains a second target. A mixture comprising the first target, the second target, a first pair of primers to amplify the first target, a second pair of primers to amplify the second target, and a nonspecific reporter that binds without specificity to an amplification product is partitioned. The method amplifies the first target and the second target in the partitions and detects an intensity of light emitted by the nonspecific reporter from individual partitions.
The invention further determines a level of each of the first and second targets by using a positive or negative count of partitions for the first or second target based on the intensity detected from the individual partition. The nonspecific reporter is used to bind without specificity to amplification product so that fluorescence intensity from individual partitions is classified as positive or negative for each target. By assigning partition populations to target combinations, partition counts are used to determine target levels or concentrations, optionally using Poisson statistics as described.
The invention also includes multiplexing without target-specific probes by using primer pairs and by controlling distinguishability of amplicons so that fluorescence intensity clusters are assigned to target combinations such as negative partitions, single-positive partitions for each target, and double-positive partitions containing both targets. The document further describes use with alternative splicing targets (GUSB, CAMTA1, TPM3, ABLIM1) and approaches to address issues including signal decay via thermal conditioning, target masking/overlap, and primer-dimer/byproducts that are distinguished from true target signals.
Claims Coverage
The independent claim covers a multiplexed digital assay that uses partitions, two target-specific primer pairs, and a nonspecific reporter whose light intensity in each partition is used to count positive/negative partitions for each target and determine target levels. The inventive features are driven by (i) partitioning with selectively populated target-containing subsets, (ii) nonspecific reporter-based intensity detection per partition, and (iii) level determination using positive/negative counts for each target. Dependent claims further specify constraints and signal relationships regarding primer selection and behavior of the nonspecific reporter.
Partition-based multiplexing with target-specific primer pairs and nonspecific reporter
Forming partitions including only a first subset containing a first target and only a second subset containing a second target, wherein the mixture comprises the first target, the second target, a first pair of primers to amplify the first target, a second pair of primers to amplify the second target, and a nonspecific reporter that binds without specificity to an amplification product; amplifying the first target and the second target in the partitions; and detecting an intensity of light emitted by the nonspecific reporter from individual partitions.
Target level determination using positive or negative partition counts based on detected intensity
Determining a level of each of the first and second targets using a positive or negative count of partitions for the first or second target based on the intensity detected from the individual partition.
Primer concentration relationship between primer pairs
At least one primer of the first primer pair is present at a lower concentration than each primer of the second primer pair.
Primer melting temperature relationship between primer pairs
At least one primer of the first primer pair has a lower melting temperature than each primer of the second primer pair.
Distinct target length differentiation
The first target has a different length than the second target.
The claim set, as provided, centers on a partitioned multiplexed digital assay using a nonspecific reporter and two target-specific primer pairs, where per-partition reporter intensity is converted into positive/negative partition counts to determine target levels, with further refinements specifying primer concentration and primer melting temperature relationships and using different target lengths for differentiation.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Alternative splicing targets (GUSB, CAMTA1, TPM3, ABLIM1).
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