Development and Characterization of A Multiplexed RT-PCR Species Specific Assay for Bovine and one for Porcine Foot-and-Mouth Disease Virus Rule-Out

Abstract

, 925-422-5545, smith350@llnl.gov Contributors:Linda Danganan, 925-422-2553, danganan1@llnl.gov Lance Tammero, PhD., 925-423-9545, tammero2@llnl.gov Beth Vitalis, PhD., 925-422-0149, vitalis1@llnl.gov Chemical and Biological Countermeasures Division Nonproliferation, Homeland and International Security (NHI)Lawrence Livermore National Laboratory (LLNL) Livermore, CA Principle Investigators: Ray Lenhoff, Ph.D., PhD., 925.422.5665, pnaraghi@llnl.gov Benjamin Hindson, PhD., hindson1@llnl.gov This work was performed under the auspices of the U.S. . This effort has the ability to improve our nation's capability to discriminate between foreign animal diseases and those that are endemic using a single assay, thereby increasing our ability to protect food and agricultural resources with a diagnostic test which could enhance the nation's capabilities for early detection of a foreign animal disease.In FY2005 A timeline for this development is presented in Table 1. The development of the Version 1.0 panel for FMDV rule-out and the most current efforts aimed to designed species specific panels has spanned over 2 ½ years with multiple collaborative partnerships.This document provides a summary of the development, testing and performance data at OIE Stage 1 Feasibility into Stage 2 Assay Development and Standardization 1 (see Table 2), gathered as of June 30 th , 2007 for the porcine and bovine MUX assay panels. We present an overview of the identification and selection of candidate genetic signatures, the assay development process, and preliminary performance data for each of the individual signatures as characterized in the multiplexed format for the porcine and bovine panels. The Stage 1 Feasibility data of the multiplexed panels is presented in this report also includes relevant data acquired from the Version 1.0 panel as supporting information where appropriate. In contrast to last years' effort, the development of the bovine and porcine panels is pending additional work to complete analytical characterization of FMDV, VESV, SVD, RPV and MCF. The signature screening process and final panel composition impacts this effort. The unique challenge presented this year was having strict predecessor limitations in completing characterization, where efforts at LLNL must precede efforts at PIADC, such challenges were alleviated in the 2006 reporting by having characterization data from the interlaboratory comparison and at Plum Island under AgDDAP project. We will present an addendum at a later date with additional data on the characterization of the porcine and bovine multiplex assays when that data is available. As a summary report, this document does not provide the details of 90% complete: during development, each MUX panel was tested against viral nucleic acid extracts (total nucleic acids, non-titered) from each organism. Preliminary results assessed by low-resolution titration curves, for each step-wise addition of assay to the multiplexed panel. 1.2.1.2.Calibration test of reference standards 90% complete: refere...