Quantification of Influenza D and Mycoplasma bovis
Influenza D virus was quantified in nasal swab, BAL fluid, and tissue samples using a one-step RT-qPCR as previously described (10 (link)). Briefly, the viral polymerase basic 1 (PB1) gene was amplified with specific primers and quantified by using a specific probe and the QuantiNova probe RT-PCR kit (Qiagen, Germany) on a LightCycler 96 real-time PCR system (Roche, Switzerland). Viral copy numbers in samples were determined by using a standard plasmid containing the PB1 product of influenza virus D/bovine/France/5920/2014 (18 (link)). For quantification of M. bovis genomic DNA copy numbers in nasal swab, BAL fluid, and tissue samples, qPCR was performed with specific primers, probe, and specific standard using the Bio-T Mycoplasma bovis PCR kit (Biosellal, France) on the LightCycler 96 real-time PCR system (Roche, Switzerland) according to the manufacturer’s instructions.
Lion A., Secula A., Rançon C., Boulesteix O., Pinard A., Deslis A., Hägglund S., Salem E., Cassard H., Näslund K., Gaudino M., Moreno A., Brocchi E., Delverdier M., Zohari S., Baranowski E., Valarcher J.F., Ducatez M.F, & Meyer G. (2021). Enhanced Pathogenesis Caused by Influenza D Virus and Mycoplasma bovis Coinfection in Calves: a Disease Severity Linked with Overexpression of IFN-γ as a Key Player of the Enhanced Innate Immune Response in Lungs. Microbiology Spectrum, 9(3), e01690-21.
Other organizations :
Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement, Université de Toulouse, École Nationale Vétérinaire de Toulouse, Centre Val de Loire, Swedish University of Agricultural Sciences, Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna "Bruno Ubertini", Swedish Veterinary Agency
Influenza D virus quantification in nasal swab, BAL fluid, and tissue samples
dependent variables
Viral copy numbers of the Influenza D virus PB1 gene
Genomic DNA copy numbers of Mycoplasma bovis
control variables
Use of specific primers and probes for the Influenza D virus PB1 gene and Mycoplasma bovis
Use of the QuantiNova probe RT-PCR kit and Bio-T Mycoplasma bovis PCR kit
Use of a standard plasmid containing the PB1 product of Influenza D virus D/bovine/France/5920/2014
Use of the LightCycler 96 real-time PCR system
controls
Positive control: Standard plasmid containing the PB1 product of Influenza D virus D/bovine/France/5920/2014
Negative control: Not explicitly mentioned
Annotations
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