Biosprint 96 nucleic acid extractor

At the National Reference Laboratory (INIAV), rabbit pathogens associated with high mortality rates, namely RHDV, RHDV2 and MYXV, were investigated. Liver samples from the 11 specimens were homogenised with phosphate buffered saline (PBS) and clarified at 3000 g for 5 min. DNA and RNA were extracted from 200 μl of the clarified supernatant, corresponding to approximately 50 mg of tissue, in a BioSprint 96 nucleic acid extractor (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Samples were tested for RHDV2 by a specific RT-qPCR [23 (link)]. Screening for RHDV [62 (link)] was performed by sequencing of the amplicons obtained with primers RC9F and RC10R [62 (link)]. Conventional RT-PCR and RT-qPCR were performed with the One Step RT-PCR kit (Qiagen, Hilden, Germany).
The presence of myxoma virus was investigated by qPCR [26 (link)], using the FastStart TaqMan Probe Master Kit (Roche, Roche Diagnostics GmbH, Manheim, Germany).
For the real-time PCR systems described, undetectable Cq or Cq values >40 were considered negative.

For nucleic acid extraction, fresh samples of liver and spleen were homogenised at 20% (w/v) with phosphate buffered saline (PBS) and clarified at 3000 g for 5 min. Total DNA and RNA were extracted from 200 μL of the clarified supernatants, using the MagAttract 96 cador Pathogen Kit (Qiagen, Hilden, Germany) in a BioSprint 96 nucleic acid extractor (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. The nucleic acids were preserved at −20 °C until use.

For nucleic acid extraction, fresh samples of liver and spleen were homogenised at 20% with phosphate buffered saline (PBS) and clarified at 3000g for 5 min. Total DNA and RNA were extracted from 200μl of the clarified supernatants, using the MagAttract 96 cador Pathogen Kit in a BioSprint 96 nucleic acid extractor (Qiagen, Hilden, Germany), according to the manufacturer’s protocol.
All the animals were tested for rabbit haemorrhagic disease virus 2 (RHDV2) and MYXV by real time PCR systems described by Duarte et al (2015) [12 (link)] and Duarte et al (2014) [22 (link)], respectively. The presence of LEHV-4 was investigated by using the PCR described by Jin et al (2008) [9 (link)]. A generalist nested PCR directed to the herpesviral DNA polymerase that allows the detection of herpesviruses of different subfamilies by Van Devanter et al. (1996) [16 ] was also used.
The glycoprotein B gene was partially amplified using the GH1 system described previously [17 (link)].
Amplifications were carried out in a Bio-Rad CFX96^™ Thermal Cycler (Bio-Rad Laboratories Srl, Redmond, USA), using the One Step RT-PCR kit (Qiagen, Hilden, Germany) for RHDV2, and the HighFidelity PCR Master Mix (Roche Diagnostics GmbH, Mannheim, Germany), for MYXV and herpesvirus detection, respectively.
Information regarding these methods is summarized in Table 2.