molecular dx

The 2PG MoMâ„¢ can detect and quantitate DNA/RNA targets with concentration levels across a broad dynamic range. If amplification is required, both isothermal and thermocycling is supported within the test strip. Most 3rd party primers and probes from existing reagents are typically supported.

The 2PG MoMâ„¢ is an open system platform that allows independent developers to create assays for molecular and other diagnostic tests. See the Partners page for more information.

Silicon nanopores: single molecule sensors

Silicon nanopore sensors electrically sense individual molecules, one at a time. No optics. No chemistry.

Voltage pulls molecules through, impeding current by a measured amount.

Matrix contains DNA, RNA, proteins, small molecules, resulting in many different event signatures. Single Molecule “Event” has depth and duration, proportional to its size.

DNA molecules are present in this sample. How do we identify the desired targets apart from background? And how can we quantitate them?

Detecting amplified genomic markers

Goal: Screen saliva sample spiked with HCV at clinical concentration levels (~500 copies per ml)

  • Swabbed sample is placed into test strip is inserted into hand-held device for in situ HCV amplification (7 mins).
  • Amplified product is detected alongside a synthetic calibrant within the well, and a mathematical analysis establishes a semi-quantitative result.
  • Data is produced and analyzed in real time
  • Encrypted data is wirelessly transmission to selected devices and databases.

Comparing detected events from a HCV positive sample (blue) to a HCV negative sample (black) shows clear detection of the positive sample above background.

Comparing HCV positive event population (blue) to a sequentially run calibrant (red), allows fractional abundance and semi-quantitation of starting HCV viral RNA concentration

Multiplexed SNP detection

Method: Multiplex estimating the fractional abundance of KRAS mutation G12D transcripts compared to KRAS wild-type transcripts (UCSF study data)

  1. Post cfDNA extraction, target sequence is amplified using minimal PCR cycles (10 mins)
  2. SNP specific probes-payloads are added and allowed to bind (5 mins)
  1. Tagged amplicons (WT and mutant) are simultaneously detected in the pore (1 min). The different payload sizes are classified by their distinct event populations for mutant (black) and wild-type (blue), with false positives (<0.5%).

Different payloads produce different impedance signatures

Assay demonstrates:

  • Multiplexing
  • SNP detection using probe-payloads
  • Fast assay time
  • PCR unbiased amplification method achieves rare target detection

Multiplexing multiple targets

Raw sample contains Chlamydia, Gonorrhea, Trichomonas (100 copies), among other background.

Amplify all DNA together in the same well. Each primer set contains a payload: 1, 2, or 3 PEGs.

Post-amplification, 100bp amplicons are tagged with different sized payloads.

PEGs cause amplicons to produce unique signal populations. Scatter plot shows each target is present in the sample, demonstrating three-plex.

Quantitative Multiplexing

  • Use primer sets to generate amplicons of different lengths to detect and quantitate multiple targets in a single test.
  • Example below assumes amplicons representing HCV, HBV, HIV, Zika and Syphilis
  • All-event histogram peaks reveals the DNA lengths present, with math framework for predicting amounts.

Assay demonstrates:

  • Each marker is amplified to a defined length in a multiplexed amplification reaction
  • Amplicons of mixed length are run simultaneously in the nanopore to determine which markers are present, and their relative amounts

Assay Sensitivity

Depending on the starting number of templates copies in a sample, amplification of nucleic acids may or may not be required. Assay developers can direct the MoM to amplify targets as needed to achieve desired test results, sensitivity levels, and time-to-results.

Amplification Free

  • Single nanopore provides results (99.5% CV) in minutes when target DNA is at low pM concentrations (pM, 1e-12).
  • Detection of low fM concentrations (fM,1e-15 M) using DNA concentration step.

Amplification

  • Detection of sub-aM concentrations (aM, 1e-18)
  • Low cycle reactions accurately quantitated in seconds, starting at 10-100 template copies.
  • Arrays of pores lowers limits of detection (and/or reduces time-to-results).

Sensitivity and Dynamic Range

2PG Molecular Dx Comparison

Single Pore 100 Pore Hybridization Chips qPCR Digital droplet PCR
Quantitative Accuracy w/in +/- 5% < 5% +/- 15% +/- 20-40% +/- 5%
Quantitative Precision w/in 1-5% < 1-5% 15% 50-10% 10%
Quantitative Range (pg/ul) 0.3 - 30,000 0.003 - 30,000 1 - 10,000 15 - 150,000 ng/ul 10 - 100,000 copies
Dynamic range 5-logs 7-logs 5-logs 4-logs 4-5-logs
Test Time Minutes Minutes 30 min to overnight Minutes-hrs 4 hours