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LLNL Awarded $2.5M to Move Pathogen Detection Tests to NanoString Platform


By Justin Petrone

The National Institutes of Health has awarded $2.5 million to researchers at Lawrence Livermore National Laboratory to support the development of pathogen detection assays on NanoString Technologies' nCounter system.

The project aims to develop high-content, multiplex assays capable of simultaneously detecting 35 National Institute of Allergy and Infectious Diseases category A, B, and C viral agents, plus quantifying cytokine and chemokine responses in patients, according to the grant abstract.

The project, called "Multiplexed diagnostic and cytokine response assays utilizing the nCounter platform," was awarded on May 1 and is set to end on April 30, 2014. The funding amount for 2011 is $1,075,661. Other partners listed on the project include NanoString, the University of Texas' Medical Branch, and the University of California, San Francisco.

Principal investigator Pejman Naraghi-Arani told BioArray News last week that he expects additional funding of about $1.5 million in 2012 and 2013 to support the validation of the assays, meaning the total funding for the project will be approximately $2.5 million.

Since joining LLNL in 2004, Naraghi-Arani has worked in biodefense, with a focus on developing multiplex assays for the detection of biothreat agents from the environment, many using RT-PCR or Luminex's xMAP technology.

"Our main focus of research has been on detection assays for detection of various A, B, and C category viruses and bacteria and we have developed over 450 TaqMan-based assays for almost all category A, B, and C agents," Naraghi-Arani said.

The list of NIAID's category A, B, and C pathogens is available here. It includes such agents as anthrax, botulism, plague, smallpox, and tularemia.

The nCounter Analysis System, sold by Seattle-based NanoString, is a newer addition to Naraghi-Arani's lab. The nCounter relies on molecular barcodes and single-molecule imaging to detect and count transcripts in a single reaction. Each color-coded barcode is attached to a single target-specific probe corresponding to a gene of interest. Mixed together with controls, the probes form a multiplexed panel that the firm calls a CodeSet.

Among the catalog CodeSets sold for use on the nCounter is NanoString's GX Human Inflammation Kit, comprising 184 human genes known to be differentially expressed in inflammation. Using the kit, customers can measure the levels of chemokines and cytokines in an individual sample, for example.

Naraghi-Arani plans to build upon the existing inflammation assay by adding amplicons currently used in LLNL's TaqMan assays. The result would be an assay that can monitor inflammation, as well as identify the causative pathogen.

"Since we have amplicons that are between 80 and 180 base pairs, we thought it would be quite simple to take the amplicons and import them into the NanoString nCounter," said Naraghi-Arani. "As cytokine and chemokine assays are already available, we could basically combine the two and have a profiling assay that tells you the special level of the chemokines and cytokines and messenger RNA, as well as viral target RNAs," he said.

Using the resulting assays, the LLNL researchers hope to be able to "assess the patient's state of health, whether or not he or she is sick, and if he or she is sick, with which one of these 35 viruses the patient is infected," Naraghi-Arani added.

It is unclear whether NanoString will eventually commercialize the assays developed at LLNL. A company spokesperson this week said that the firm is "excited" about the project, but declined to further elaborate.

According to the grant abstract, the envisioned assay will require minimal sample preparation with no need for amplification or enzymatic reactions and will allow the detection of up to 700 analytes per sample.

An initial throughput of up to 72 samples per instrument per 24-hour period is planned. As custom CodeSets can be ordered directly from NanoString, modifications to the assay can be made as needed, so that the system can integrate new tests for other agents and other host-response genes.

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The flexibility to design CodeSets as warranted is one of the reasons that Naraghi-Arani decided to build the new diagnostics on the nCounter platform as opposed to other technologies, such as microarrays, but it wasn't the only one.

"We felt arrays wouldn't be as informative and would be more labor-intensive and they would be more expensive," he said.

LLNL researchers have developed a pathogen-detection array, however. The Lawrence Livermore Microbial Detection Array, referred to as LLMDA, contains probes representing about 178,000 viral sequences from some 5,700 viruses, and about 785,000 bacterial sequences from thousands of bacteria. The latest version of the chip, which is manufactured by Roche NimbleGen, became available last year (BAN 5/10/2010).

Naraghi-Arani, who was not involved in the development of LLMDA, said the array-based system is suited for pathogen discovery, but would not be effective as a diagnostic tool. "You have to do an upfront, whole-genome amplification and then you have to do a hybridization," Naraghi-Arani said of the array. "So, basically, you get no time savings and you have more work to do for yourself," he said. "And each array costs about $650, whereas each nCounter [assay] costs about $350," Naraghi-Arani noted.

He said that he had evaluated other technologies, such as Fluidigm's integrated fluidic circuit-based systems, but again decided that the nCounter was the appropriate platform for this project.

Naraghi-Arani added that he has opted to move other projects to the nCounter platform for similar reasons. For instance, he initially used microRNA arrays for oncogene discovery in a project last year, but later decided to move the targets identified to the nCounter for validation.

Have topics you'd like to see covered in BioArray News? Contact the editor at jpetrone [at] genomeweb [.] com