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Image by Solen Feyissa
Hyperplex PCR Technology

PCR Supercharged

Our technology has unlocked the true potential of molecular analysis.

We have taken multiplexing for biomarker detection to a whole new level. But not only that, our molecular counting assay enables unprecedented capabilities of specificity, sensitivity and quantification. 

Under the hood

These are the technologies supercharging Hyperplex PCR

Multiplex unleashed

The Nanopixels are novel fluorescent nanoprobes invented and developed by Aplex Bio. Compared to regular fluorophores, they are brighter, more photostable and have unique fluorescent properties making them capable of generating 100+ colors using only 4-6 fluorescense channels. The surface of the Nanopixel is carefully optimized to enable high precision DNA hybridization and they are labelled with universal sequences enabling plug-and-play compatibility with any custom panel without the need for optimization.

The workflow

01 PCR & RCA amplification

The sample is amplified in a 2-step process. The first a conventional multiplex PCR amplification, followed by a rolling-circle amplification (RCA) in any standard thermocycler.

02 Capture & Nanopixel labelling

The amplified sample is captured on a specialized microscope slide using a multisample frame, followed by simultaneous labelling with 100+ colors of Nanopixels. The Nanopixels have universal tags and only bind to their target-specific barcoded RCP while unbound nanopixels are washed away. This enables an unprecedented level of multiplexing in each well with efficient workflow.

03 Imaging & automated image analysis

The microscope slide is imaged using a conventional epi-fluorescense microscope with 4-6 fluorescence channels. The images are analyzed in our fully automated analysis software outputting molecular counts for each target based on Nanopixel color recognition and counting, leading to 100+ plex capabilities per well. Continue reading about what molecular counting means.

How the Hyperplex PCR assay works

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01 PCR amplification

 

Extracted sample containing ssDNA, dsDNA and RNA undergoe a conventional multiplex PCR amplification. The number of cycles is adjusted depending on input concentrations and sensitivity need, enabling up to single-molecule sensitivity.  Primer design is simple since specificity is introduced in the next step.

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02 Padlock-probe ligation

 

The amplicons are then probed by padlock-probes (PLPs) to introduce specificity and filtering of potential PCR artifacts. High-fidelity ligation mediated circularization is achieved only upon perfect sequence match, giving the ability to discriminate mutations with single-nucleotide specificity and eliminate false-positives from PCR artifacts. A mismatch does not get circularized and therefore not amplified in the next step.

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03 Rollining-circle amplification

 

The circularized PLPs containing the target region together with the designated Nanopixel barcode are  amplified by a linear amplification process called rolling-circle amplfication (RCA) to generate micrometer sized RCA products (RCPs) containing multiple copies of the encoded Nanopixel barcode. The RCPs are bright enough to be counted individually, enabling molecular counting.

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04 Nanopixel labelling

 

All 100+ Nanopixels are added to the sample simultaneously and only the barcode specific Nanopixels bind to their target RCPs. The Nanopixels are multiple fold brigther than regular fluorophores, photostable and capable of generating more than 100 colors using only 4-6 standard fluorescence channels. Multiple Nanopixels of each color bind their target RCP creating a strong signal able to be detected by fluorescence microscopy. This self-assembly of arrays enables unprecedented multiplexing in each well without the need of sample splitting, complex consumables or sequencing.

Molecular counting - what it means

In conventional dPCR, input molecules are partitioned by physical confinement leading to the unavoidable consequence of (1) multiple molecules in one partition giving an ambiguous and unpure signal and (2) several empty and unused partitions.

With rolling-circle amplification, each molecule is elongated into a long strand containing thousands of repeats forming a micrometer sized DNA microball called rolling-circle product (RCP) leading to a pure signal from each RCP.

Advantages:

  • Signal from RCP is clean without the ambiguities of mixed molecule partitions in conventional dPCR.

  • Pure signal enables unparallelled multiplexing combined with Nanopixels.

  • Each RCP represents an originating molecule, enabling digital quantification by molecular counting.

  • Millions to billions of RCPs are generated in each sample, leading to virtually unlimited counts.

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The RCPs appear as bright fluorescent spots in an image, each representing a single DNA target molecule. Our automated image analysis software counts each spot, reads its Nanopixel color and translates it into its corresponding target - giving an intrinsically digital read-out without the need for compartmentalization as in digital PCR.

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Different targets within one sample can be precisely quantified relative to each other, such as mutant vs wild-type sequences, a target AMR gene vs 16S rRNA reference or the frequency of mutations vs a conserved gene in a viral genome. Using molecular counts coupled with appropriate internal references provides unparalleled quantification precision.

High counts for high multiplex

1M+ counts

More than 1 million useful molecular counts can be analyzed per well depending on imaging area and sample concentration. Compared to conventional dPCR solutions, this is a 100-1000 fold improvement enabling unparallelled precision in analysis. In order to achieve true hyperplex, it is essential to secure the necessary counts not to compromise accuracy of the analysis.

In this microscopy image, approximately 250k counts were acquired.

Click image to zoom

1M Counts
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