Lightcycler rna amplification kit hybridisation probes

Total RNA from serum, nasal swabs and probang samples were isolated using the MagNA Pure 96 DNA and Viral NA Large Volume kit, on the MagNA Pure 96 system (Roche® Life Science). In each run of 96 samples, one negative, one high positive and one moderate positive sample were included as extraction controls. The RT-PCR was carried out as described by the manufacturer (Roche®), using the LightCycler RNA Amplification Kit Hybridisation Probes and LightCycler 480 (Roche® Life Science), using the protocol described by Moonen et al. [28 (link)]. In each run of 96 samples, one negative, one high positive and one moderate positive RT-PCR control were included. The test protocol was as follows: reverse transcription for 20 min at 61 °C, denaturation for 1 min at 95 °C, followed by 45 PCR cycles of 3 s at 95 °C, 15 s at 60 °C and 10 s at 72 °C. Amplification was monitored in real time, using hybridization probes. Samples were considered positive when the fluorescence signal rose above the background signal (crossing point determined automatically by the second derivative maximum method for quantification by the software supplied by Roche®) [28 (link)].

Viraemia (serum) and virus excretion in oral and nasal swabs and probang samples were studied by virus titration and real-time RT-PCR. Primary ovine kidney cells supplied by the WBVR were used to perform virus isolation and titration using a standard plaque assay, and the results expressed as log₁₀ PFU/mL [23 (link)]. Samples were recorded as negative when no plaques were observed. As the plates were washed and stained, a second passage was not possible. Total RNA from 200 µL of serum, oral and nasal secretions (as mentioned above) and probang samples were isolated using the MagNA Pure 96 DNA and Viral NA Large Volume kit on the MagNA Pure 96 system (Roche^® Life Science) and real-time RT-PCR was carried out using the LightCycler RNA Amplification Kit Hybridisation Probes and LightCycler 480 (Roche^® Life Science) using the protocol described by Moonen et al. 2003 [24 (link)]. Samples were declared positive when the fluorescence signal rose above the background signal (crossing point determined automatically by the second derivative maximum method for quantification by the software supplied by Roche^®).