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Digital PCR

Digital PCR (dPCR) is a powerful tool used for the precise and highly sensitive quantification of nucleic acids. As the third generation PCR technology, digital PCR is uniquely capable of absolute quantification.

The concept of dPCR is to divide a sample into tens of thousands of parts and distribute them within different reaction units, each unit contains at most one or few target molecules (DNA template). In each reaction unit, the target molecule is subjected to PCR amplification, and finally statistical analysis by fluorescent signal via the Poisson Distribution. This is a calculation of the absolute number of DNA molecules in the sample which can be detected.

Digital PCR is increasingly multiplexed. By targeting multiple sequences simultaneously, multiple results can be obtained from a single run that would otherwise require several tests, and consequentially, higher reagent input and more time to get the results of all the experiments combined. The increase in multiplexity has found a home in translational genomics applications such as in oncology, organ transplant, and infectious diseases screening.

Top Applications for Digital PCR

6-color naica® system workflow

Stilla Technologies is taking digital PCR to a whole new level. The 6-color naica® system is a complete digital PCR solution with flexible sample throughput, and a unique 6-color target detection capability which enables another exclusive dimension for multiplexed dPCR assays while maintaining an easy-to-use workflow.

Digital PCR enables several types of assays, allowing multiplexing

Absolute Quantification (DNA/RNA) Copy Number Variation (CNV)
Rare Event Detection Gene Expression
Liquid Biopsies (CTC, cfDNA) Droplet Recovery
Whole Genome Amplification (WGA) Imaging Droplets pre/post-PCR or at any time during reaction (stop and start)
NGS Library Calibration / NGS Results Validation
Single Cell Analysis

Crystal Digital PCR

An advanced digital PCR equipment, the Naica System, was launched in 2016 by Stilla Technologies. This system performs digital PCR by partitioning the sample, using a confinement gradient into a large 2D array of droplets, also called a droplet crystal.

A PCR reaction occurs in each of the partitioned 25,500-30,000 droplets that make up the droplet crystal, and a fluorescence read-out is performed at end point by taking high-resolution images of the crystal. This digital PCR workflow, named Crystal™ Digital PCR, combines the advantages of a) array-based digital PCR such as an integrated workflow and multiplexing; with b) those of droplet-based PCR such as reduced cost.

The ability to partition the sample extensively and uniformly affords the system with superior precision and sensitivity for target detection and quantification. Additionally, the flexibility to explore different fluorophores, the capability of performing multiplexing of targets (up to 6- colors), and a fast time to result make it a unique digital PCR system.

Highly multiplexed design through up to 6 fluorescent channels

The accurate and precise quantification of multiple targets in a single reaction saves you both time and precious sample. The naica® system gives researchers the ability to read up to six fluorescent signatures in one well, providing better sensitivity and increased low-level detection of multiple reactions in parallel.

EGFR Detection using the EGFR 6-color Crystal Digital PCR kit

  • The EGFR 6-color Crystal Digital PCR™ kit reliably detects 32 common, rare, activating, and resistant somatic EGFR mutations in a single assay.
  • The EGFR 6-color Crystal Digital PCR™ kit is optimized for ultrasensitive and highly robust EGFR mutation detection on the naica® system with LoDs ranging from 0.30 to 0.46 cp/μL, depending on the target using Sapphire chips.
  • The Crystal Miner software and custom EGFR 6-color analysis template enable fast and straightforward analysis of cfDNA.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer mortality worldwide. Epidermal growth factor receptor (EGFR) is frequently mutated and is a well known genetic aberration in NSCLC. The highly multiplexed, ready-to-use EGFR 6-color Crystal Digital PCR™ kit allows the detection from circulating free DNA (cfDNA) of more than 90% of EGFR mutations described in NSCLC, including 32 common, rare, activating, and resistant somatic EGFR mutations in exons 18, 19, 20 and 21.

Stilla® Technologies’ 6-color naica® system is an ultrasensitive digital PCR technology capable of simultaneously and precisely quantifying high numbers of biomarkers in a single sample, simplifying the detection process, minimizing detection variability, and significantly reducing the hands-on-time to results.

EGFR 6-color Crystal Digital PCR™ kit detection design. TaqMan™ probe positions on each exon and fluorescent color code are noted

LoB and LoD of the EGFR 6-color Crystal Digital PCR™ kit determined following the CLSI EP17-A2 standard (Sapphire chip well concentrations in cp/μL)

Target LoB95 LoD95
DEL19+ 0.11 0.37
L858R 0.09 0.38
INS20+ 0.17 0.46
L861Q, G719X 0.09 0.37
T790M 0.12 0.46
C797S 0.06 0.30

A representative example of the analysis of a L858R and T790M positive NSCLC clinical cfDNA sample using Crystal Miner software and the custom EGFR 6-color Crystal Digital PCR™ kit analysis template. The cfDNA sample showed a perfect concordance between the expected and measured results. The x- and y-axis correspond to the fluorescence units of the indicated color channel.

Environmental Monitoring

Wastewater epidemiology has been a highly researched topic during Covid-19. Digital PCR systems have been widely used to detect trace amounts of SARS-CoV2 in wastewater, owing to its ability to differentiate target of interest DNA and undesired byproducts.

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