Digital Pcr Assay
Digital Pcr Assay - Related Videos
12:14
A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters
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Cited by 14 •
2016
This manuscript describes a duplex digital PCR assay that can be used to simultaneously quantify Enterococcus spp. and the HF183 genetic markers as indicators of general and human-associated fecal contamination in recreational waters.
08:45
Duplex Digital PCR for Simultaneous Quantification of Dual Genetic Markers
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2025
In this video, we demonstrate the duplex digital PCR (ddPCR) technique — a modification of the traditional PCR technique that is useful in detecting two different genetic markers simultaneously. A single PCR reaction is partitioned into nanoliter-sized emulsified droplets that are independently amplified, and the detection of differently colored fluorescence amplification signals from the fraction of droplets is used to compute the initial concentration of the target sequences.
07:37
Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR
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Cited by 10 •
2016
A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.
04:32
Chip-in-a-Tube-Based Digital PCR for Quantification of Single Nucleotide Variants
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2025
This video demonstrates the utility of digital PCR in a chip-in-a-tube format for the detection of single nucleotide variations in DNA samples. A single PCR reaction is partitioned into chambers that act as independent PCR reactions, and the detection of fluorescence signals from the amplified targets in the chambers is used to compute the frequency of the variant allele in the sample.
06:55
Simple Bulk Readout of Digital Nucleic Acid Quantification Assays
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Cited by 5 •
2015
We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.
FAQs
Poisson statistics correct for the possibility that a partition received more than one target molecule. By comparing the numbers of fluorescently positive and negative partitions, the analysis estimates the original concentration from the pattern of occupancy rather than from signal intensity alone. This calculation allows copy estimates to remain absolute and avoids dependence on a calibration curve.
Endpoint fluorescence provides a binary readout for each partition: target-containing reactions are classified as positive, while reactions without detectable target are classified as negative. Counting these categories supports statistical estimation after amplification, so the assay can distinguish small differences in target abundance even when overall sample concentrations are low.
Compared with conventional quantitative PCR, digital PCR does not require a calibration curve to convert amplification measurements into copy estimates. That distinction is particularly useful when the expected difference is small or when sample complexity makes comparative quantification difficult. The result is a direct count-based measurement rather than an estimate tied to external standards.
A typical workflow starts by preparing a DNA or complementary DNA sample, distributing it across many individual reaction partitions, and completing endpoint amplification. Fluorescence then separates positive from negative partitions, after which Poisson-based analysis converts those counts into a starting-copy estimate. Each stage contributes to the final measurement, so consistent partitioning and signal classification are important.
Its tolerance of inhibitors can preserve measurement performance in samples that interfere with conventional quantitative PCR. That feature matters when the sample matrix is complex, because inhibition may otherwise compromise amplification-based detection. In practice, this makes a digital PCR assay useful for biological research and testing contexts where sample composition is less controlled.
Applications span rare-mutation detection, copy-number analysis, pathogen detection, and quantification of low-abundance transcripts. Within biological techniques, the method is valuable when researchers need sensitive, reproducible measurements from challenging samples. The same capabilities also support diagnostic and environmental testing, extending its relevance beyond routine molecular experiments.