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LLNL Scientists Develop Microbial Detection Array for Biodefense, Forensics Use

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By Justin Petrone

Researchers at Lawrence Livermore National Laboratory in California have developed a microarray-based microbial-detection platform that they claim can be used to identify viruses and bacteria for a number of applications.

The scientists detailed the use of the array combined with a metagenomic sequencing approach in a recent Journal of Virology paper. Working with San Francisco's Blood Systems Research Institute, the team first sequenced eight vaccine samples and then used the chip to confirm the presence of porcine circovirus-1, a highly prevalent non-pathogenic pig virus, in Rotarix, an orally administered human rotavirus vaccine manufactured by GlaxoSmithKline.

BSRI notified GSK of the finding, and GSK subsequently informed the US Food and Drug Administration that Rotarix contained PCV1. Based on the new information, the FDA in March recommended that healthcare practitioners temporarily suspend use of Rotarix while the agency learned more about PCV1.

Last week, the FDA revised its recommendation, and now believes clinicians can continue to use Rotarix after finding no evidence that PCV1 poses a safety risk in humans.

Crystal Jaing, a biologist at LLNL, told BioArray News last week that a research team at BRSI led by Eric Delwart first identified the presence of PCV1 in Rotarix after sequencing the sample with the Roche 454 GS FLX system, but wanted to validate the finding using another technology.

Delwart "identified the pig virus but wanted a second validation because it was a very controversial finding," Jaing said. "By using sequencing and arrays, we were able to identify what was in the product."

The Lawrence Livermore Microbial Detection Array, referred to as LLMDA, was initially developed in the lab of LLNL investigator Tom Slezak in 2007, Jaing said. A prototype was ready for use in early studies in 2008, and a second version of the array, which includes 388,000 probes for the detection of more than 2,000 viruses and 900 bacteria, became available last year.

Jaing said that LLNL is now implementing a third generation of the LLMDA, which will be manufactured by Roche NimbleGen and contain 2.1 million probes. Previous generations of the arrays were manufactured in-house at LLNL using NimbleGen technology.

The new version of the 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 LLMDA also includes fungi and protozoa, with probes representing about 237,000 fungal sequences from thousands of fungi and about 202,000 protozoa sequences from 75 protozoa, Jaing said. She added that the array is now being tested and the analysis software is being optimized.

The LLNL team has said it plans to update probes on the array with new sequences of bacteria, viruses, and other microorganisms from GenBank and other public databases about once per year, in addition to using sequences obtained from collaborators for their probes.

"With PCR kits, you can do only one pathogen at a time," she said in explaining the motivation for developing the LLMDA. "We wanted to design a comprehensive array that could be used in clinical diagnostics, biodefense, and product safety, so you could tell what's in your sample."

Jaing said that LLNL is interested in licensing the technology and is involved in collaborations with other research groups. For example, Jaing said that the lab is working with the Marine Mammal Center in Sausalito, Calif., in an effort to identify what may be a viral basis for cancer in seals and sea lions at the center. To do that, LLNL has obtained carcinoma tissue samples for array analysis to identify all viruses present.

LLNL is also working with the US Department of Homeland Security's National Biodefense Analysis and Countermeasures Center on bioforensic projects, Jaing said. "They are planning to bring our array technology into operations," she confirmed.

Other collaborators include the University of Texas Medical Branch in Galveston; the National Institute for Public Health and the Environment of Bilthoven, the Netherlands; the Statens Serum Institut of Copenhagen, Denmark; the University of California, Davis; and St. Petersburg, Fla.-based bacterial infection detection firm Imigene.

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