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Sandia Researchers Developing Multiplex Assay Differentiating Viral Febrile Illnesses, Malaria

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NEW YORK (GenomeWeb) – Researchers at Sandia National Laboratories have been awarded a grant to develop a field-deployable assay for differential diagnosis of malaria and viral febrile illness.

The two-year grant from the National Institute of Allergy and Infectious Disease totals $188,759 and is expected to fund the proof-of-concept phase of the project. The Sandia research team will also be collaborating with researchers at the Institute for Human Health and Immunity at the University of Texas Medical Branch in Galveston.

The point-of-care molecular diagnostic device will potentially discriminate infection from a panel of viruses causing febrile illness as well as the parasite causing malaria.

Specifically, the proposed technology will use closed-tube isothermal nucleic acid amplification to detect and discriminate viruses endemic in West Africa — Ebola, Lassa, yellow fever, chikungunya, dengue, and West Nile viruses — as well as Plasmodium falciparum.

The amplification method of choice is reverse-transcription loop-mediated isothermal amplification (RT-LAMP), Robert Meagher, lead researcher on the project at Sandia, told GenomeWeb in an interview.

Although multiplex LAMP has been reported in the literature — a BioTechniques study in 2012, for example — Meagher said the method has not yet been widely adopted. The Sandia technique is also different, and has some unique advantages, particularly in that the multiplexing is enabled by improvements the group has made in visualization.

While many using LAMP rely on turbidity or add an intercalating dye like SYBR Green, Meagher said his group's strategy is more akin to a fluorophore and quencher-based approach.

"That adds an element of target specificity and allows us to do multiplex detection," he explained.

The group has a manuscript in revision describing the detection chemistry which shows multiplex detection of West Nile and Chikungunya virus, Meagher said.

All of the viruses selected have RNA-based genomes. Plasmodium's intercellular RNA can be developed as the target, but DNA polymerase is also included in the RT-LAMP reaction, so the group could choose to target the parasite's DNA-based genome instead, he said.

A number of the viruses in the panel also require Biosafety Level-4 containment. Initially, the work will use inactivated material from collaborator Scott Weaver at UTMB Galveston National Lab.

"If this work progresses and shows promise, our hope would be to test with live infectious virus, and those experiments could be conducted in the BSL-4 lab," Meagher said.

The platform will use volumes in the microliter range, so at scales more similar to a PCR tube than a microfluidic device, Meagher said. It will use liquid sample to hydrate dried reagents in the cartridge.

So far in the lab, the assays work on diluted whole blood, but the device may also incorporate some element of streamlined RNA extraction.

Meagher has previously co-authored research in Biomicrofluidics into a droplet-based microfluidic system that can encapsulate analytes, as well as studies in Lab on a Chip on solvent replenishment for digital microfluidics, as reported by GenomeWeb, and picoinjection. He also co-authored the study describing the Sandia microfluidic next-generation sequencing library prep system, which allows users to process small numbers of DNA samples in an automated fashion.

The group has filed a provisional patent for the RT-LAMP detection chemistry. There are also elements of the microfluidic device that it is currently pursuing IP on, Meagher said.

In terms of industry relationships, Meagher said the group is getting to the point where it might be looking for partners interested in making use of its LAMP detection chemistry.

"We haven't gone out actively searching yet, but as we're getting our IP filed and getting our manuscript … closer to publication I think we'd be interested in partnering with people."

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