Magattract 96 cador pathogen kit

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.

The CSFV RNA were extracted from sera, nasal and rectal swabs, and tissues from vPdR-H₃₀K-5U-infected pigs by the MagAttract 96 cador pathogen kit (Qiagen), according to the manufacturer’s instructions. Tissue samples were ground in sterile Eagle’s minimal essential medium (EMEM; 1 g of tissue plus 9 mL of EMEM) supplemented with 2% penicillin (10,000 U/mL) and streptomycin (10,000 U/mL). RT-qPCR (48 (link)) was used to quantify the CSFV RNA in the collected samples. C_T values equal or lower than 40 were considered positive. Samples that did not show any detectable fluorescence were considered negative. As previously quantified, C_T values from 10 to 23 were defined as high, from 23 to 29 as moderate, and between 29 and 40 as low RNA viral loads (16 (link), 49 (link)).

CSFV RNA was extracted from sera, nasal, and rectal swabs, and tonsils. The tonsil tissue was homogenized in Eagles MEM (1 g tissue +9 ml medium) supplemented with penicillin 10,000 U/ml, streptomycin 10,000 µg/ml, and used for CSFV RNA detection. The MagAttract 96 cador Pathogen Kit (Qiagen) was used for RNA extraction following the manufacturer’s instructions, and the RNA was stored at −80°C until CSFV RNA analysis by quantitative reverse transcription-PCR (RT-qPCR) [32 (link)]. Cycle threshold (Ct) values of 40 and below were considered as positive. Ct values between 29 and 40 were considered as low, from 23 to 28 as moderate and from 10 to 22 as high viral RNA load as previously [33 (link)].

Tissue samples were homogenized in Eagles sterile Minimal Essential Medium (EMEM; 1 g of tissue + 9 mL EMEM) 6% supplemented with penicillin 10,000 Units/mL, streptomycin 10,000 U/mL, and nystatin antibiotics 10,000 U/mL, and tested for Pestivirus RNA detection. RNA was extracted using the MagAttract 96 cador Pathogen Kit (Qiagen), following the manufacturer’s instructions. In all cases, extraction was performed from an initial sample volume of 200 μL to obtain a final volume of 100 μL of RNA, which was stored at −80 °C. The presence of Pestivirus RNA in the blood, serum, tissues, and in nasal and rectal swabs was analyzed by the quantitative reverse transcription-PCR (RT-qPCR) [21 (link)] and CSFV-specific RT-qPCR [22 (link)]. Threshold cycle (Ct) values equal to or lower than 40 were considered as positive. Samples in which fluorescence was undetectable were considered as negative. As previously described, Ct values from 10 to 23 were considered as high, from 23 to 29 as moderate, and between 29 and 42 as low RNA viral load [23 (link)]. Positive RT-PCR samples in tissue samples were inoculated into cell cultures for virus isolation in SFT-R cells by peroxidase linked assay (PLA) test.

Nucleic acid extraction was performed by accredited means within the collaborating laboratories: i) MagNA Pure Compact (Total Nucleic Acid Extraction Kit, input volume of 200μl or 400μl and an elution volume of 50μl) and MagNA Pure 96 (DNA and Viral NA Small Volume Kit, input volume of 200μl and an elution volume of 50μl) extractors (all from Roche), ii) Nucleomag Vet kit (Macherey-Nagel) or MagAttract 96 cador Pathogen Kit (Qiagen) on King Fisher Flex 96 (Thermo Fisher Scientific): Input volume 200μl and elution volume 100μl, and iii) manual and robotic extraction depending on the origin of the specimen, as described by Slomka et al. [48 (link)].

Viral RNA was extracted from all the serum, tissue macerate, and swab samples from all pigs, using the MagAttract 96 cador Pathogen Kit (Qiagen), from an initial sample volume of 200 µL. Tissue samples had been previously ground in 900 µL of Eagle’s Minimum Essential Medium, with the supernatant being used for extraction after centrifugation at 13,000 RPM for 10 min. After extraction, the RNA was stored at −80 °C, until analysis by the specific pan-CSFV RT-qPCR test [33 (link)], as well as the specific Margarita strain RT-qPCR [34 (link)]. Reactions were performed in a final volume of 20 µL, using the AgPath-ID™ One-Step RT-PCR Reagents (applied biosystems, Waltham, MA, USA): RT-PCR buffer (1x), RT-PCR Enzyme mix (1x), forward primer (0.6 µM), reverse primer (0.6 µM), probe (0.1 µM) and RNA template (4 µL). The temperature profile was 10 min at 48 °C, 10 min at 95 °C, followed by 40 cycles of 2 s at 97 °C and 30 s at 61 °C. Samples were considered positive when cycle threshold (Ct) values were below 40. Samples were also characterized as having high (Ct below 22), moderate (Ct between 23 and 28) and low (Ct between 29 and 40) viral RNA load, as previously described [31 (link),35 (link)].