Automated Extraction and RT-PCR for BTV

BTV RNA was extracted from 100 μl of EDTA blood using the KingFisher Flex automated extraction platform (ThermoFisher Scientific, Paisley, UK) and the MagVet Universal nucleic acid extraction kit (ThermoFisher) and RNA was eluted into 80 μl. Neat EDTA blood samples were extracted in triplicate while each of the pooled EDTA blood samples (1:2, 1:5, 1:10 and 1:20) were extracted and tested in ten replicates. Five microliters of BTV RNA was denatured at 95 °C for 5 min prior to analysis using two different real-time RT-PCR assays. The VetMAX assay was used according to the manufacturer’s instructions. The Hofmann et al., (2008) (link) assay was performed using 15 μl of the reaction mix using the Express One-Step Superscript qRT-PCR kit (LifeTechnologies, Paisley, UK) containing 1 × reaction mix, 400 nM forward and reverse primers, 200 nM probe, 0.5 μl Rox, and 2 μl of enzyme mix in each well. Cycling conditions were as follows: reverse transcription at 50 °C for 15 min and 95 °C for 20 s min, and then 45 cycles of PCR, with each cycle consisting of 95 °C for 3 s, 56 °C for 30 s and 72 °C for 1 min. Real-time RT-PCR was performed on an Applied Biosystems 7500 Fast instrument (LifeTechnologies) using the fast ramp rates to provide results within 90 min for both assays.