Viral Genome Load Analysis in Animal Samples

Lymph node samples as well as skin samples were homogenized in serum-free medium (FLI cell culture medium ZB5d, containing MEM (H), MEM (E) + non-essential amino acids) with antibiotics using the TissueLyser II tissue homogenizer (QIAGEN, Hilden, Germany). For DNA extraction of all samples of both animal trials, the KingFisher Flex System (Thermo Scientific, Darmstadt, Germany) was used with the NucleoMag Vet kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. An internal control DNA (IC-2 DNA) was added during the DNA extraction process for control of successful DNA extraction and inhibition free amplification [56 (link)]. For analyses of viral genome loads in all samples, the already described pan-Capripox real-time qPCR [57 (link)] with a modified probe [58 (link)] was performed using the PerfeCTa qPCR ToughMix (Quanta BioSciences, Gaithersburg, MD, USA).

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Wolff J., Moritz T., Schlottau K., Hoffmann D., Beer M, & Hoffmann B. (2020). Development of a Safe and Highly Efficient Inactivated Vaccine Candidate against Lumpy Skin Disease Virus. Vaccines, 9(1), 4.

Publication 2020

TissueLyser II tissue homogenizer KingFisher Flex System NucleoMag Vet kit PerfeCTa qPCR ToughMix

Animal Antibiotics Cell Cell culture Culture medium Essential amino acids Inhibition Lymph node Serum Skin Tissue Viral genome

Corresponding Organization : Friedrich-Loeffler-Institut

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Variable analysis

independent variables

Homogenization of lymph node samples and skin samples in serum-free medium (FLI cell culture medium ZB5d, containing MEM (H), MEM (E) + non-essential amino acids) with antibiotics using the TissueLyser II tissue homogenizer

dependent variables

Viral genome loads in all samples

control variables

Addition of an internal control DNA (IC-2 DNA) during the DNA extraction process for control of successful DNA extraction and inhibition free amplification

controls

Positive control: Internal control DNA (IC-2 DNA) added during DNA extraction
Negative control: Not explicitly mentioned

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