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Seattle Children's Developing Test for Emerging Flu Strains on Sony Micronics PanNAT Platform

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Researchers from Seattle Children's Hospital, working with Sony subsidiary Micronics, are developing a point-of-care diagnostic to identify and track new influenza strains under a five-year, $5.3 million grant from the National Institute of Allergy and Infectious Diseases.

The test will adapt flu assays using a method called consensus-degenerate hybrid oligonucleotide primer (CODEHOP)-based PCR — developed by the Seattle Children's group and used mainly over the last decade to detect novel viruses in primates — and marry them to PanNAT, a microfluidic molecular diagnostics platform under development at Micronics.

Timothy Rose, the project's principle investigator, told PCR Insider this week that the group hopes the resulting POC test will be a tool for more sensitive and specific monitoring of emerging pandemic influenza strains by the US government.

Rose noted that the government and the US Centers for Disease Control want to be able to quickly diagnose new pandemic strains of influenza. However, "existing systems are targeted to known viruses and may be able to say if you have a novel virus, but if four different novel viruses come in, you can't really track them individually," Rose said.

The team's CODEHOP-based PCR will allow for a test that not only detects new viruses, but also recognizes the unique signature of a new virus so that it can be tracked individually.

"If a particular new strain pops up once, it's not a problem. But if you start seeing 10, 100,1,000 of the same new virus, the idea is that that information would be fed back to the CDC as an advanced warning from these POC devices," he explained.

Most of Rose and colleagues' previous work with CODEHOP PCR has been focused on chronic viruses like herpes and papillomavirus. But after seeing a presentation on Micronics' platform at a conference, Rose said he approached the company to try to identify something that would be a promising commercial target to adapt CODEHOP to the PanNAT device, settling on respiratory viruses.

Rose then began early research on a number of respiratory viruses, leading most recently to the effort to commercialize a first flu test product under this new grant, Rose said.

The newly announced NIAID funding, which began earlier this month and is worth approximately $1.2 million in its first year, will support adaptation of the Micronics platform to be able to run CODEHOP flu assays, Rose said. The end goal is an integrated test combining nasal swab extraction, cDNA conversion, amplicon detection, and discrimination, which the group will hopefully validate through prospective testing of clinical samples through several flu seasons.

Micronics, which Sony acquired in 2011, has been developing PanNAT as its cornerstone technology: a lightweight, portable, integrated testing platform using nucleic acid amplification as its core detection technology.

According to the NIAID grant abstract, funding for the flu test effort will cover completion of the PanNAT instrument and cartridges, "including instrument modification for melt curve analysis and analytical performance testing with standardized synthetic plasmids and clinical samples."

At the same time, the Seattle Children's researchers will be making adaptations to their assays in order to move them out of a laboratory PCR setting and marry them to the PanNAT microfluidic POC device.

Rose's CODEHOP-based PCR, which he and his colleagues first published in 1998 in Nucleic Acids Research, utilizes a primer design that allows amplification of unknown targets by targeting a conserved amino acid sequence.

According to the team's publication on the method, CODEHOP primers consists of a short 3' degenerate core region representing three to four highly conserved amino acid residues coupled to a longer 5' consensus clamp region that stabilizes the core during annealing to template molecules.

"If you are trying to identify an unknown gene or an unknown pathogen you can look at the conservation of amino acid sequences of a protein from different members of that family to find regions where the protein sequences are absolutely conserved, which means you'd expect an unknown pathogen in that family to have the same sequence," Rose explained.

"Since the amino acid sequence defines, to some extent, the underlying nucleotide sequence, you can use that information to predict what the nucleotide sequence would be of an unknown target," he said.

Because amino acids can have different numbers of codons, Rose said the group tries to pick conserved motifs where the amino acids have just one or two codons. "Then we can make a pool of primers that have a small amount of degeneracy — maybe 16 primers in a pool that will give every single nucleotide combination encoding those three to four amino acids," he said.

The "consensus" part of the method refers to the added non-degenerate 5' sequence in each primer. During initial PCR amplification cycles, the degenerate core region is responsible for specific binding to sequences encoding the conserved amino acid motif. The longer consensus clamp stabilizes the primer and allows the participation of all primers in the pool in the amplification of products during later PCR cycles.

"We've found that this approach is much better than typical approaches like completely degenerate oligos, where there can be mismatches throughout the whole length of the primer," Rose added.

Over the next five years under the new NIAID funding, Rose said his team hopes to be able to test the CODEHOP-PanNAT test over a number of flu seasons, using samples from patients at Seattle Children's, to collect real-world evidence that the test can not only detect the presence of new strains, but also distinguish them so that they can be tracked individually.

When the group can start this work will depend on how long the process of integrating CODEHOP PCR with Micronics' platform takes. "In the grant we thought it would happen early, but it looks like it may take a little more time," he said.

According to the grant abstract for the project, successful development of the Flu-CODEHOP assay will also give a leg up for future diagnostic applications for Micronics' PanNAT platform, potentially for "a wide range of respiratory and enteric pathogens or other agents that pose a biological threat to humans."

Micronics declined to comment on other diagnostic applications it is pursuing for PanNAT, or how it expects the CODEHOP flu test effort with Rose and his colleagues to help advance the platform.

Rose said though, that Micronics' POC system is unlikely to be viable unless it hosts a number of different assays.

"With the system we developed with CODEHOP, we can identify any gene based on its relation to known genes," Rose said. "So that's something I was really excited about, that with this system you could develop assays to go against a whole variety of pathogens."

"We haven’t talked much with [Micronics] about the future of that," he said. "But the idea is that if this works, they could develop more assays [using this approach] down the line."

Micronics has received previous funding — $2.6 million in 2010 from the US Department of Defense — to develop molecular tests on the PanNAT system to directly detect hepatitis B and C and HIV from fresh blood samples in order to screen blood donated for transfusion on the battlefield.

The company has also said that it is developing tests for enterohemorrhagic Escherichia coli, various respiratory infections, and mother-to-child transmission of HIV.

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