Perfecta qpcr toughmix

For the homogenization of organ samples in a serum-free medium, the TissueLyzer II tissue homogenizer (Qiagen, Hilden, Germany) was used. Subsequently, the DNA of all samples taken during the study was extracted using the KingFisher Flex System (Thermo Scientific, Darmstadt, Germany) and the NucleoMag Vet kit (Macherey-Nage, Düren, Germany) according to the manufacturer’s instructions. During DNA extraction, an internal control-DNA (IC-2 DNA) was added to the samples to control successful DNA extraction and inhibition-free amplification [44 (link),45 (link)]. The analysis of the viral genome load in the samples was performed using the PerfeCTa qPCR ToughMix (Quanta BioSciences, Gaithersburg, MD, USA) and the already described pan capripox real-time qPCR of Bowden et al. [6 (link)] with a modified probe [46 (link)].

The air samples collected in the disposable sample chambers were stored at room temperature until the conclusion of the study and transported under standard shipping conditions. All samples were analysed in the AeroCollect laboratories at FORCE Technology as blinded samples.
The collected material in the disposable sample chambers were eluted by pipetting in 25 µL Mili-Q water. Each sample was analysed in duplicate according to the procedure, primer and probes originally described by Josefsen, et al.^{31 (link)}. Briefly, 2 µL template from the eluted samples were analysed in a total volume of 20 µL containing 10 µL qPCR master mix (PerfeCTa^® qPCR ToughMix^®, Quanta Biosciences Inc., Beverly, MA, USA) probe, forward and reverse primers at a final concentration of 0.05 µM, 0.44 µM and 0.48 µM, respectively. Water was added to reach the final concentration of 20 µL. The reactions were performed in an Agilent Aria Mx qPCR (Agilent Technologies, Inc., Santa Clara, CA, USA). After 3 min at 95 °C, 40 cycles were performed at 94 °C for 20 s and 58 °C for 60 s, respectively. The detection limit is between 0.3 and 3 genomic copies/µl.

An equimolar quantity of VIC-labelled mGAPDH primers and probes (Applied Biosystems, assay no. 4352339E) was added as an internal control into each keratin qPCR. RNA was reverse-transcribed according to the manufacturer's protocol (High Capacity Reverse Transcription kit, Life Technologies). Krt12/KRT12 qPCR reactions (perfeCTa^® qPCR toughmix; Quanta Biosciences, Gaithersburg, MD, USA) contained cDNA equivalent to 1.25 ng/µl total RNA. qPCR reactions for other keratins (TaqMan Universal Mastermix II, Life Technologies) contained cDNA equivalent to 1.8 ng/µl. A TaqMan 7900HT machine (Applied Biosystems Paisley, UK) was used for quantification of triplicate samples using the ΔΔCt method (77 (link)).

Total bacterial/archaeal small sub-unit (SSU) rRNA gene count was estimated using a TaqMan based probe assay previously designed to provide even and accurate amplification of bacteria and archaea within a sample (Liu et al., 2012 (link)). Briefly, the assay was carried out in 25 μL reactions containing 1x final concentration of PerfeCTa qPCR ToughMix (Quanta BioSciences Inc.), 1.8 μM of each primer, and the probe at a final concentration of 225 nM using samples collected and extracted in July 2017 as template for each reaction. Each biological replicate was also assayed in technical triplicate. A seven-point standard curve was generated using serially diluted genomic E. coli DNA in triplicate.

First, all organ samples were homogenized in serum-free medium using the TissueLyser II tissue homogenizer (QIAGEN, Hilden, Germany). DNA extraction from all samples taken during the animal trial (EDTA blood, serum, nasal swabs), as well, as the homogenized organ samples was performed with 100 mL sample material using the NucleoMag Vet kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions on the KingFisher Flex System (Thermo Scientific, Darmstadt, Germany). The extracted nucleic acid was eluated in 100 µL elution buffer. For control of successful DNA extraction, an internal control DNA (IC-2 DNA) was added during the extraction process [29 (link)]. Afterwards, a pan Capripox real-time qPCR using the PerfeCTa qPCR ToughMix (Quanta BioSciences, Gaithersburg, MD, USA) was performed according to the protocols of Bowden et al. (primer) [3 (link)] and Dietze et al. (probe) [30 (link)] using 2.5 µL template DNA in a 12.5 µL qPCR total volume.

Organ samples were homogenized in a serum-free medium using the TissueLyser II tissue homogenizer (Qiagen, Hilden, Germany). The extraction of homogenized tissue samples and samples taken during the animal trial was performed using the NucleoMag Vet kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions, with volume modifications as described before [48 (link)], utilizing the KingFisher Flex System (Thermo Scientific, Darmstadt, Germany). During the extraction process, an internal control (IC-2 DNA) was added for control of successful DNA extraction and inhibition-free amplification [49 (link)]. For an analysis of the capripox virus genome, the pan capripox real-time qPCR described by Bowden et al. [50 (link)] with a modified probe of Dietze et al. [51 (link)] was performed utilizing the PerfeCTa qPCR ToughMix (Quanta BioSciences, Gaithersburg, MD, USA).