Qtower3 g qpcr cycler

DNA from the Auckland Island pigs was used to analyze the presence of PERV-C using a conventional PCR. Primers and conditions used were described previously as PCR1 [22 (link),23 (link)] (Table 1). In addition, a real-time PCR was established using specific primers and probe (Table 1) [23 (link)]. 100 ng DNA and the SensiFAST Probe No-ROX kit (Meridian Bioscience Cincinnati, Newtown, OH, USA) in a 20 µL reaction volume. The cycling conditions used were initial denaturation for 5 min at 95 °C, followed by 45 amplification cycles of 95 °C for 15 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s in a qTOWER3 G qPCR cycler (Analytik Jena, Jena, Germany). A standard curve was produced using the 510 bp amplicon of previously described PCR6 [23 (link)] (Table 1) as template.
This PCR was performed as follows: 100 ng of DNA template, PCR buffer I containing MgCl₂, 0.2 mM dNTPs, and 1 unit of AmpliTaq DNA polymerase (Applied Biosystems, Inc., Waltham, MA, USA). As positive control, DNA from the PERV-C positive pig #6249) was used. Each sample was subjected to an initial denaturation of 10 min at 95 °C, followed by 45 amplification cycles (95 °C for 15 s, 58 °C for 30 s, and 72 °C for 40 s) and a final extension at 72 °C for 5 min The sensitivity of the real-time PCR was 10 copies (Supplemental Figure S1).

Real-time PCRs were performed to detect PCMV/PRV, PLHV-1, PLHV-2, PLHV-3, PCV2, PCV3, PCV4 and PPV1 as described previously using specific primers and probes (Table 1) [16 (link),17 (link),18 (link),19 (link),20 (link),21 (link),22 (link)]. All protocols were performed using the SensiFAST Probe No-ROX kit (Meridian Bioscience, Cincinnati, OH, USA) in a reaction volume of 16 µL plus 4 µL (100 ng) of DNA template. All real-time PCRs were carried out as duplex PCRs that simultaneously indicate the gene of interest and porcine glyceraldehyde-3-phosphate-dehydrogenase (pGAPDH) as internal control for each sample. Real-time PCR reactions were carried out with a qTOWER3 G qPCR cycler (Analytik Jena, Jena, Germany) and the real-time PCR conditions as previously described [16 (link)].

Real-time reverse transcriptase-PCR (real-time RT-PCR) as described by Jothikumar et al. [23 (link)] was carried out to detect hepatitis E virus, genotype 3 (HEV3). All real-time RT-PCR reactions were performed in a reaction volume of 16 µL using SensiFAST Probe No-ROX One-Step Kit (Meridian Bioscience, Cincinnati, OH, USA) plus 4 µL (100 ng) template RNA. The reaction was performed at the qTOWER³ G qPCR cycler (Analytik Jena, Jena, Germany). The temperature-time profile applied consists of a reverse transcriptase step of 30 min at 50 °C, followed by an activation step of 15 min at 95 °C and 45 cycles comprising a step of 10 s at 95 °C, followed by a step of 20 s at 55 °C and 15 s at 72 °C [24 (link)].