Nucleospin virus core kit

Nucleic acid extraction from serum and tissue samples (lung tissue, pulmonary lymph nodes and tonsillar scrapings) was conducted using the Nucleospin® Virus Core kit and the Nucleospin 96® RNA kit (Macherey-Nagel, GenXpress, Wiener Neudorf, Austria), respectively, on the automated platform Freedom EVO® 150 (Tecan, Grödig, Austria), following the instructions of the manufacturer.
To detect PRRSV 1 and 2 RNA, the samples were analysed by a commercial ORF7 RT-qPCR assay that allows the simultaneous detection and differentiation of PRRSV 1 and 2 (TaqMan® PRRSV Reagents and Controls, Life Technologies, Brunn am Gebirge, Austria) on the ABI 7500 Fast Real-Time PCR System (Life Technologies). For absolute quantification, a PRRSV 2 RNA dilution series with known copy numbers ranging from 1.0E + 00–1.0E + 07 copies/μl was assayed in parallel.

Total RNA was extracted and purified from cell culture supernatants or mosquito bodies using the NucleoSpin virus core kit (Macherey-Nagel) following the manufacturer’s instructions with RNA elution in 50 μl of RNase-free water at 70°C for bodies and 100 μl for cell culture supernatants. cDNA synthesis was performed using M-MLV reverse transcriptase (Invitrogen) by mixing 5 μl of eluted RNA with 100 ng of random primers (Roche), 10 nmol of each deoxynucleoside triphosphate, 2 μl of dithiothreitol, 4 μl of 5× first-strand buffer, 5.5 μl of PCR-grade water, 20 units of RNaseOUT recombinant RNase inhibitor (Invitrogen), and 200 units of M-MLV reverse transcriptase in a final reaction volume of 20 μl. The reaction mixtures were incubated for 10 min at 25°C, 50 min at 37°C, and 15 min at 70°C and held at 4°C until further use or stored at −20°C. Diagnostic PCRs for all three viruses was performed with DreamTaq Green DNA polymerase (Thermo Scientific) following the manufacturer’s recommendations. Quantitative analysis by qPCR was done using GoTaq qPCR master mix (Promega) following the manufacturer’s recommendations. The primer sequences are provided in Table 1.

PCV3 DNA was detected using a real-time PCR assay published previously by Palinski and colleagues [2 (link)]. First, serum pools (n = 5) were screened for PCV3. If a sample pool was PCV3 DNA positive, the included single samples were tested subsequently. If two or more serum pools from one farm were tested positive, only the pool with the lowest Cq (quantification cycle) value (indicating highest viral load) was investigated further. In total, we tested 212 serum pools and 200 single samples.
DNA from serum samples was isolated using the NucleoSpin Virus Core Kit (Macherey-Nagel) and a Microlab Starlet workstation (Hamilton Robotics) according to the manufacturer’s instruction. For the real-time PCR we used the QuantiTect probe PCR kit (Qiagen). Oligonucleotide primers (PCV3-F: 5′ AGT GCT CCC CAT TGA ACG; PCV3-R: ACA CAG CCG TTA CTT CAC) were used with a final concentration of 800 nM, the TaqMan probe (5’ FAM-ACC CCA TGG CTC AAC ACA TAT GAC C-BHQ1) was used with 200 nM. The thermal profile of the PCR was: 94 °C for 15 min, and 42 cycles of 94 °C for 15 s and 60 °C for 60 s.