NEW YORK – Sweden-based startup Aplex Bio has seemingly set a record for most targets in a single PCR reaction with its Hyperplex PCR kits.
Officially launched on Friday, the firm's chemistry multiplexes up to 100 targets in a single tube by combining padlock probes, rolling circle amplification, and a fluorescent intensity-based readout using universal nanoparticle probes.
Aplex Bio CEO Umear Naseem said in an interview that the chemistry has been extensively validated in independent wastewater epidemiology and forensics labs in Sweden, demonstrating high sensitivity and specificity for highly multiplexed assays even in samples notoriously riddled with PCR inhibitors.
Accompanying the launch, Aplex Bio released a white paper describing its technology. The technical demonstration targeted 50 human indels of one to three base pairs using 100 padlock probes and 50 primer pairs with amplicon lengths ranging from 107 to 170 base pairs, tested using synthetic ssDNA targets.
In the technical demonstration, hpPCR had accuracy exceeding 99.96 percent, mutation detection below 0.1 percent frequency, and quantification across more than four orders of magnitude.
Although padlock probes and RCA have been combined before — namely by Mats Nilsson, a scientific adviser to Aplex Bio and researcher at the Swedish national research center SciLifeLab — the addition of nanoparticle probes, dubbed Nanopixels, is what enables the high multiplexing, Naseem said.
These universal probes can "generate many different nuances of color" by modulating the intensity with high precision, Naseem said, akin to pixels on a television monitor. This approach essentially expands the color space of the probes to 100 distinct, quantifiable codes, he said.
Similar to digital PCR, the method can essentially isolate each nucleic acid being amplified. But unlike dPCR, the compartmentalization is not physical, Naseem said, but rather, is "a molecular compartmentalization."
Specifically, the combination of padlock probes and RCA spools the nanoparticle probes into a 1-micron ball at the site of amplification.
With these so-called rolling circle products, "we are actually counting each target DNA molecule," Naseem said.
Furthermore, the method has out-of-the-box single-nucleotide specificity, Naseem said, while its sensitivity is enhanced by its generation of more than 1 million molecular compartments.
By comparison, standard droplet-based PCR generates roughly 20,000 droplets, although startup Enumerix has developed a dPCR method to generate 30 million droplets and enhance dynamic range, albeit with a more limited level of multiplexing.
Indeed, as it is neither precisely real-time PCR nor digital PCR, Aplex Bio's high-plex PCR technology arguably necessitates a new analytical methodology moniker: hpPCR.
However, although each hpPCR reaction essentially generates a bead-like blob of fluorescence that can be counted automatically with imaging software, similar to dPCR, absolute quantification of hpPCR still requires generation of a standard curve, Naseem said.
Based at the Karolinska Institute in Stockholm, Aplex is currently part of the KI Innovations incubator.
Early adopters of hpPCR include researchers at the Swedish National Forensic Centre who are now also further testing the method in collaboration with the Netherlands Forensic Institute.
The Swedish Environmental Epidemiology Centre, meanwhile, recently published results of a one-year study of hpPCR-based SARS-CoV-2 variant tracking in wastewater, benchmarking the technology against next-generation sequencing and qPCR. This group has now expanded its evaluation to include a respiratory surveillance panel for wastewater that supplies data to the Swedish Public Health Agency.
And, at the University of Gothenburg, researchers have used hpPCR for gene expression analysis of food crops, showing strong concordance with qPCR.
Going forward, Nassem said Aplex Bio intends to serve customers in the applied markets represented by these early users, as well as research and clinical customers who may require large molecular panels for in vitro diagnostics and precision medicine.
And, in the long term, Aplex also aspires to develop regulated molecular panels, Naseem said.
Aplex is offering the hpPCR chemistry as customizable kits in 20-, 50-, and 100-plex configurations. And, if 100-plex isn't enough, the technology also enables detection beyond 100 targets using clustering strategies to associate multiple targets with each code.
The hpPCR reactions use standard thermocyclers and off-the-shelf imaging systems or epifluorescence microscopes, with results read out as images that are automatically analyzed using the accompanying software. Aplex Bio also plans to release optimized protocols later this year to pair the kits with liquid handling systems, and to launch an integrated liquid handling and imaging system in the next few years.
Along with the kits, the firm is marketing consumables available as either microscope slides or 96- and 384-well plate formats amenable to high throughput automation. Using hpPCR, labs can process up to four 384-well plates per day, with each sample having 100-plex capability.
The demonstration panel presented in its white paper was also an example of Aplex's in-development Human ID forensic assay that aims to replace short tandem repeat (STR) analysis with indels in a single-tube reaction.
Multiplexing is increasingly becoming vital to molecular testing. Naseem cited Bio-Rad Laboratories' acquisition of Stilla Technologies as an example of an industry player racing to increase multiplexing capabilities and reduce instrument complexity while facing growing competition — in this case, from dPCR offerings by Qiagen, Thermo Fisher Scientific, and Roche.
Offerings from Luminex, ChromaCode, InBiome, Agena Bioscience, and Seegene, as well as a 96-plex research assay called MeltArray, have a similar flavor and aspirations in terms of multiplexed molecular testing, but Naseem said these technologies haven't managed the same high level of multiplexing paired with high accuracy as hpPCR.
Aplex Bio has so far raised $3.3 million in financing, Naseem also said, and the firm is now planning a fundraising round in the near future to help it grow its sales.