For the entire validation process, nucleic acids were isolated using the QIAsymphony DSP Virus/Pathogen Midi kit (Qiagen, Hilden, Germany) on a QIAsymphony SP instrument (Qiagen, Hilden, Germany) (sample volume 300 μL; custom protocol), unless otherwise specified. During the lysis phase, all the samples were spiked with an exogenous internal control (intype IC-RNA, Indical Bioscience GmbH, Leipzig, Germany) to reproduce routine laboratory conditions as foreseen by the upstream AIV screening method adopted at the IZSVe [60 (link),61 (link)] that employs the same nucleic acids.
Amplification reaction was assembled with the AgPath-ID One-Step RT-PCR Reagents (Applied Biosystems, Waltham, MA, USA), 400 nM primer for, 200 nM each primer rev, 200 nM probe, 20 units RNase inhibitor and 5 μL template, in a final volume of 25 μL. Thermal cycling was performed on a CFX96 Deep Well Real-Time PCR System, C1000 Touch (Biorad, Hercules, CA, USA), as follows: 50 °C for 10 min, 95 °C for 10 min, followed by 45 cycles at 95 °C for 15 s, 54 °C for 30 s and 72 °C for 15 s. Data were analyzed using Bio-Rad CFX Manager software (Version 3.1) (Biorad, Hercules, CA, USA), with fluorescence drift correction for the baseline adjustment and single threshold manually set above the background noise (c.ca 50 RFU).
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