NEW YORK – Countable Labs, formerly known as Enumerix, expects its chemistries and benchtop reader will allow researchers to measure single DNA molecules quickly and cheaply.
After recently rebranding, the Palo Alto, California-based startup will launch its first product, called Countable PCR, at the American Society of Gene & Cell Therapy meeting next week.
The firm has developed a method to create 30 million tiny compartments within a gel matrix in a standard PCR strip tube, all within about 20 minutes.
When applied to PCR reactions, the approach is similar to digital PCR but obviates the need for standard curves or Poisson statistics.
"The simplest way to measure something is counting, and that's what we're doing," said Jocelyn Davé, the firm's new CEO. Although the firm's first application for its technology is PCR, Countable plans to expand into other assays. "We consider our technology essentially like a picoliter bioreactor."
Eleen Shum, Countable's founding scientist and VP of product development, said the Countable approach, along with its simple workflow and automated results, can potentially speed up scientific projects, at a lower cost than standard methods.
For example, the 30 million compartments allow researchers to do their work using fewer total reactions, she said. The approach also yields error bars with a CV of approximately 1 percent, a 10-fold improvement over digital PCR. The approach has no dead volume or sample loss, as it uses a centrifugation-based method rather than microfluidics.
Countable has also developed a novel multiplexing chemistry called Universal Multiplexing, which Davé said is 10 times less expensive than standard approaches. The technique was described in a BioRxiv publication two years ago, along with a proof-of-concept panel enabling simultaneous detection of 22 pathogen targets.
Combining Universal Multiplexing with Countable PCR's dynamic range of up to 1 million molecules could also potentially shorten project times for diagnostics developers by allowing them to detect both rare and abundant targets in a single reaction with minimal optimization.
In addition to the proprietary gel matrix, the method uses its own consumables and reagents, including a spin column and PCR mix, with the PCR reaction processed on any standard thermal cycler.
Readout is done on the firm's own instrument, which can process between one and 96 samples, with four color channels. It relies on a 3D-scanning technique called light sheet microscopy, which essentially performs optical z-sectioning of a sample.
In a poster at ASGCT, the Countable team also highlighted ways its tech may be more versatile and approachable for sensitive and low-variability applications — such as copy number analysis and viral titer characterization — compared to legacy methods.
For this application, qPCR may be too variable, while dPCR can suffer from a narrow dynamic range, high Poisson errors, and arbitrary thresholding, the firm said. In addition, conducting both viral titer and genome integrity assessments in a single assay is challenging using dPCR due to a relatively smaller number of partitions or droplets.
In contrast, in its ASGCT poster, Countable demonstrated a novel multiplex assay that interrogates different regions of a gene of interest to quantify the ratio of partial and complete transgenes while simultaneously measuring viral titer.
Unlike dPCR, the single-molecule counting approach "prevents false co-occupancy of fragmented, unlinked targets, ensuring accurate genome integrity data," according to the poster. Full genome integration can be further validated with long-read amplicon analysis, as the firm demonstrated up to 2,000 bp length.
The Countable team also presented two posters at the Association for Molecular Pathology meeting last November.
The first poster demonstrated the technologies' capability to "expand the haystack," Shum said, by counting molecules from 3 μg of DNA. This method could be particularly useful for assays using tissue or whole blood samples.
The second poster showed how Countable PCR could increase confidence in rare molecule detection, which the firm suggested makes it ideal for cell-free DNA, rare cells in cerebrospinal fluid, or other low-input samples.
Early user insights
Rick Tarleton, a cell biologist at the University of Georgia, learned about Countable Labs from its proof-of-concept study in Analytical Chemistry. In his efforts to develop a diagnostic for Chagas disease, he had been searching for a way to perform many PCR assays as quickly and cheaply as possible.
Chagas disease is caused by the protozoon Trypanosoma cruzi. The parasite is transmitted in saliva from the bite of a reduviid bug — also known as the kissing bug because it commonly bites the face — and it is most common in rural, impoverished areas of South America. Chronic T. cruzi infections involving the heart and digestive system can be ultimately fatal.
Tarleton and his team at the Center for Tropical and Emerging Global Diseases are now using Countable PCR for ultrasensitive detection and quantification of T. cruzi.
In a recent publication, they described a method to perform several hundred standard PCR detection reactions to increase low-level detection of the parasite in host insects. "This approach works well, but is laborious and expensive," Tarleton said, so the team has now been working with Countable Labs instead.
According to Tarleton, the major advantages of the Countable method are the high DNA loading per reaction compared to standard PCR and the wide dynamic range. "For hosts with this infection, there is a greater than millionfold difference in parasite load," he said, but the Countable Labs method "covers this range with a single DNA input."
The team is also impressed with the multiplexing capability and hopes that using multiple targets will improve sensitivity, as well, Tarleton said.
Although his team has been focused on the Chagas disease application, Tarleton expects to have other experimental applications for the Countable system in the future, such as detection of T. cruzi in animals and humans. They are hoping to ultimately establish a clinical diagnostic lab that can offer the test for other research studies. "This is an extremely important need that is not being met by any other technology," he said.
Regarding federal funding cuts impacting global public health research, Tarleton said his team was fortunate to get in under the wire with new NIH funding for developing an assay on the Countable Labs system, allowing them to continue the project for the time being. "Unfortunately, the recent changes in policy involving foreign [collaborators] on NIH grants is going to make it difficult to demonstrate the broad utility of this approach for human infections," he said, since they are more common in Central and South America.
Countable Labs plans to install a new system in Tarleton's lab next month, Shum said, and will continue to collaborate with him to develop a high-sensitivity test for pathogen detection.
Going forward, Countable aims to also address the oncology research market, as well as clinical applications.
"We hope to grow and enable more applications," Davé said, since the technology's sensitivity "can enable more testing, earlier testing, and even more routine testing, as well."