VALIDATION OF NEW HIGHLY SENSITIVE AND ACCURATE LYOPHILIZED REAL-TIME RT-PCR TESTS FOR EARLY DETECTION OF AVIAN INFLUENZA.
SESSION ? – PETRAUSKENE, O
VALIDATION OF NEW HIGHLY SENSITIVE AND ACCURATE LYOPHILIZED REAL-TIME RT-PCR TESTS FOR EARLY DETECTION OF AVIAN INFLUENZA.
Collaboration between Applied Biosystems and Veterinary Laboratories Agency (U.K.)
Olga V. Petrauskene1, Michael Schumaker1, Catherine Fearnley2, Sueh-Ning Liew1, Yvonne R Thorstenson1, Manohar R. Furtado1, Philip Wakeley2 and Marek J. Slomka2
1 Applied Biosystems, Foster City, USA
2 Veterinary Laboratories Agency, UK
Accurate and rapid detection of avian influenza virus (AIV) promotes an immediate response and enhanced surveillance of outbreaks of disease. The Veterinary Laboratories Agency (VLA), a UK government agency specializing in animal disease surveillance and veterinary research and Applied Biosystems have collaboratively designed and validated three new Real-time RT-PCR avian influenza detection tests. The validated M-gene test detects all AIV subtypes and the H5 and H7 specific tests can discriminate the notifiable AIV subtypes H5 and H7 (Eastern hemisphere lineages). The goal of the VLA and Applied Biosystems collaboration was to make these tests more readily available for early detection of AIV in a wide range of countries and as such the assays are presented in an “easy-to-use” lyophilized bead format containing an internal positive control to monitor inhibitors in the reaction. Fifty-four AIV cultured isolates covering 11 H types and 44 positive swabs from an outbreak of AI (H5N1) in turkeys were used to determine analytical performance and diagnostic sensitivity of the assays. The lyophilized Real-time PCR assays were demonstrated to be more sensitive than the wet assays being able to detect 4 molecules of synthetic target RNA compared to 16 molecules for the corresponding wet assay. The diagnostic sensitivity of the lyophylised M-gene assay was determined to be 97.7% (43/44) whereas concurrent testing of these samples with the wet assay was only 86.3% sensitive (38/44). Using a panel of 19 non-influenza respiratory and enteric pathogens (viral and bacterial) the analytical specificity of the M-gene assay was shown to be 100%. High diagnostic specificity of the assays was also confirmed by testing 496 negative swab samples from a combination of wild bird species and poultry. The new assays are designed to rapidly and reliably detect a broad range of AIV strains in different sample types. These tests will be available in Europe and other relevant geographic regions, such as Asia and Africa. Pre-packaging of the lyophilized bead tests in “ready-to-use” PCR tubes greatly facilitates and standardizes global testing for AIV.
