Qiacube ht automated

The 10-g cheese slices were treated as described by Zago et al. [18 (link)]. Briefly, the samples were homogenized twice in a Stomacher 400 Circulator (Seward Laboratory, London, UK) with sterile sodium citrate (NaCt 2% w/v, pH 7.5). The cheese homogenate was centrifuged (14,400× g for 7 min at 4 °C) for fat removing. The pellet was then resuspended in Triton X-100 (2.5% v/v), washed twice with phosphate-buffered saline (pH 7.5), and centrifuged (8700× g, 7 min, 4 °C). Finally, after the pellet was resuspended in Tris-EDTA (0.1 M, pH 8), total dsDNA was extracted using a QIAcube HT automated station (Qiagen, Milan, Italy) using QIAamp 96 QIAcube HT kit (Qiagen, Milano, Italy). Total dsDNA was quantified fluorometrically (Qubit^TM, Life Technologies, Monza, Italy).

Swabs from hamsters were collected as described above. RNA was extracted (140 µl) using the QIAamp Viral RNA Kit (Qiagen) using QIAcube HT automated system (Qiagen) according to the manufacturer’s instructions. Sub-genomic (sg) viral RNA was detected by qRT-PCR [12 ]. Five μl RNA (150 µl elution volume) was tested with TaqMan™ Fast Virus One-Step Master Mix (Applied Biosystems) using QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems) according to the manufacturer’s instructions. Copy numbers/ml was calculated based on RNA standards.

Viral nucleic acid was extracted from 20%(v/v) fecal extract using QIAmp 96 Virus QIAcube HT kit on the QIAcube HT automated platform (Qiagen, Germany) according to manufacturer’s instructions.