Magvet universal nucleic acid extraction kit

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.

BTV RNA was extracted from 100 μl of EDTA blood and eluted into 80 μl buffer using the KingFisher Flex automated extraction platform and the MagVet Universal nucleic acid extraction kit (ThermoFisher Scientific, Paisley, UK). Ten microliters of sample RNA was analyzed as per the assay described by (Hofmann et al., 2008) with modifications (Flannery et al., 2018) using the Express One‐Step qRT‐PCR kit (ThermoFisher) on an Applied Biosystems 7500 Fast instrument (ThermoFisher). A log‐dilution series of the plasmid (1 × 10⁰–1 × 10⁶ copies per μl) was included in triplicate on each RT‐qPCR run. BTV RNA copies were determined by comparing sample C_T values to the standard curves and expressed as log₁₀ genome copies/ml.

Automated extraction of PPRV RNA was performed using 100 μl of sample (EDTA blood, cell culture isolates, tissue extracts, ocular or nasal swabs) on the Kingfisher Flex automated extraction platform (ThermoFisher Scientific, Paisley, UK) and the MagVet Universal nucleic acid extraction kit (ThermoFisher). RNA was eluted into 80 μl of elution buffer and was stored at 4 °C prior to analysis using the real-time RT-PCR assay or the RT-LAMP assay.
Manual extraction of PPRV RNA was performed on 100 μl of EDTA blood, ocular and nasal swabs using the magnetic separation rack MagnaRack (ThermoFisher) and the MagMAX CORE Nucleic Acid Purification Kit (ThermoFisher). PPRV RNA extracted from swabs was eluted into 80 μl, whereas RNA extracted from EDTA bloods was eluted into 120 μl of elution buffer. RNA was stored at 4 °C prior to analysis using the RT-LAMP assay.