Background

Agricultural pathogens can be devastating to large numbers of livestock used for meat and dairy production, having a profound negative impact on the agricultural industry. Specifically, instances of the infamous Foot-and-Mouth disease (FMD) in the past have caused tens of billions of dollars' worth of damage by prompting the infected livestock to be euthanized. Like most diseases, proper mitigation of the impact of FMD relies on the early and accurate detection of it. Current diagnostic virus isolation assays, however, may take up to a week to produce results, when countless new livestock could be infected by the highly contagious FMD virus in the meantime. Thus, the need for a timely and precise diagnostic assay is apparent.

Description

LLNL scientists have developed a high-confidence, real-time multiplexed reverse transcriptase PCR (RT-PCR) rule-out assay for foot and mouth disease virus (FMDV). It utilizes RT-PCR to amplify both DNA and RNA viruses in a single assay to detect FMDV as well as rule out other viruses that cause symptoms in livestock indistinguishable from those caused by FMDV, such as Bovine Herpes Virus-1 (BHV-1, 2), Bovine Papular Stomatitis Virus (BPSV), Bovine Viral Diarrhea Virus (BVD), Bluetongue Virus (BTV), Swine Vesicular Disease Virus (SVD) and Vesicular Exanthema of Swine Virus (VESV). This multiplexed assay contains individual Luminex-based assays for each of those seven diseases. Each assay involves probe sequences that are complementary to the target pathogen nucleic acid. The target pathogen nucleic acid is amplified via PCR, attached to the probe, and eventually tagged with a fluorescent reporter molecule for visualization by a flow cytometer. Different wavelength lasers in the cytometer analyze each final probe, producing the fast and accurate diagnostic and rule-out result.

Image: A Luminex-based assay as used in LLNL's multiplexed assay. (a) 100 unique latex microbeads, (b) individual bead with attached probe complementary to target pathogen nucleic acid, (c) PCR product pathogen nucleic acids attach to marked probe (the "+" sign), (d) the fluorescent molecule is bound, and (e) final fluorescently tagged probe is read by a flow cytometer.

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Advantages
  • LLNL's high-confidence, real-time multiplexed RT-PCR assay results would allow mitigation action to be taken early, before the disease spreads to catastrophic proportions
  • This assay would improve mitigation of FMD and similar diseases, saving billions of dollars in agricultural losses
  • One multiplexed assay, rather than a battery of tests, can be used to not only determine the presence of FMD but also to rule out six other similar diseases
Potential Applications
  • Enable agricultural labs and farmers to better mitigate the effects of FMD and other similar diseases in their region
  • Rule out "look-alike" diseases with similar phenotypic manifestations to FMD
  • This LLNL technology could be applied to assay other DNA or RNA viruses in both diagnostic and rule-out capacities
Development Status

US Patents 7,794,938 and 8,354,514 cover these technologies (LLNL internal cases IL-11578 and IL-12312, respectively)

Reference Number
43619