Ppp master mix

Reverse transcription PCR was performed using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific, #K1622, Waltham, MA, USA) and 500 ng of total RNA in a 20 μL reverse transcription reaction volume. Combined oligo (dT) and random hexamer primers were used, each with a final concentration of 2.5 μM, to prime the reverse transcription for both mRNAs and lncRNAs. Reverse transcription and quality control PCR were performed using the T100 PCR system (Bio-Rad Laboratories Inc., Hercules, CA, USA). Quality and purity (absence of DNA contamination) of the cDNA were measured by PCR using the PPP MasterMix (Top-Bio, s.r.o., Prague, Czech Republic) with GAPDH primers from the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific Inc.). Control PCR was conducted for 40 cycles in 10 μL reactions using 5 μL of PPP MasterMix, 3.5 μL molecular water and 0.5 μL GAPDH primer mix with 1 μL of sample or reverse transcriptase negative control.

RNA extracts were reverse-transcribed by a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher) using random primers according to the manufacturer’s instructions. Subsequent PCR analysis (Table S3) was performed with Phire Hot Start II DNA Polymerase (Thermo Fisher) or PPP Master Mix (Top-Bio, Vestec, Czech Republic). The quality of the cDNA was tested by amplifying a part of the Malus domestica actin gene. The PCR products were evaluated by gel electrophoresis. PCR products intended for Sanger sequencing were amplified with Q5 DNA polymerase (New England Biolabs, UK) and then cut and purified from the agarose gel using a GeneJET Gel Extraction Kit (Thermo Fisher). The products were Sanger-sequenced directly or cloned into the CloneJET vector (Thermo Fisher). Sequences of the PCR products or the cloned viruses were deposited in NCBI GenBank (Table S4).

From each test group, 40 fish were euthanized by a tricaine overdose, decapitated and dissected 3 months post-transplantation (BW: 5.6 ± 2.3 g). Firstly, gonads were visually inspected for signs of development under a light microscope. Subsequently, gonads were excised and stored separately at -80°C until RNA isolation. RNA was isolated using TRIzol reagent according to manufacturer instruction (Invitrogen). Isolated RNA was transcribed into cDNA using Transcriptor High Fidelity cDNA Synthesis Kit (Roche). Primers for RT-PCR were designed for carp and goldfish ddx4 gene (vasa) and dnd1 gene, tested for specificity and to find suitable annealing temperature (Table 1). Primers were diluted according to the manufacturer's instruction. The reaction mixture for PCR contained 1 μl template cDNA, 0.5 μl forward and 0.5 μl reverse primer, 5 μl PPP Master Mix (Top-Bio) and 3 μl PCR H₂O (Top-Bio). Reaction conditions were 35 cycles of 94°C for 30 s, 58°C (for dnd1 primers) or 60°C (for vasa primers) for 30 s and 72°C for 30 (or 45 s for vasa Carp primer) s. Products were analyzed on gel electrophoresis on 2% agarose gel on a UV illuminator.

Each RNA extract, amounting to half a microgram, was subjected to reverse transcription by a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) with random primers according to the manufacturer’s instructions. PCR (Table 1) was performed using either Phire Hot Start II DNA Polymerase (Thermo Fisher Scientific) or PPP Master Mix (Top-Bio, Vestec, Czech Republic).
The Malus domestica NADH dehydrogenase’s mRNA was used as an internal control for RNA extraction and cDNA amplification. The PCR products were analysed by agarose gel electrophoresis. Selected products were directly Sanger sequenced. If the direct sequencing was of poor quality, then the product was cloned into pJET1.2 cloning vector (Thermo Fisher Scientific), and the resulting pDNA constructs were sequenced. All obtained sequences were deposited into the NCBI GenBank (cultivar ‘Jonagored Supra’: AHVd—ON564295, SnIV-1—OR137985; cultivar ‘Selena’: AHVd—ON564296, SnIV-1— OR137986; cultivar ‘Šampion’: AHVd—ON564299).

Total RNA was isolated from the cultured cells using TRIreagent® (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer’s instructions. Reverse transcription was performed with SuperScript™ IV Reverse Transcriptase (Thermo Fisher) according to the manufacturer’s instructions, including RNaseOUT™ Recombinant Ribonuclease Inhibitor (Thermo Fisher) and Random Hexamer Primer (Thermo Fisher). RT-PCR was performed by PPP Master Mix (Top-Bio, Vestec, Czech Republic) according to the manufacturer’s instructions. Quantitative PCR was performed by using HOT FIREPol® EvaGreen® qPCR Mix Plus (Solis BioDyne, Tartu, Estonia) according to the manufacturer’s instructions and measured by LightCyler480 (Roche, Basel, Switzerland). Gapdh and Actb were used as housekeeping genes. The primers are detailed in Supplementary Table S1.

Suspect Campylobacter spp. colonies (n = 3) on blood agar were identified by multiplex PCR and matrix‐assisted laser desorption/ionisation time of flight mass spectrometry (MALDI‐TOF/MS) (Strakova et al., 2021 (link)). Briefly, bacterial DNA was extracted by thermal lysis and PCR was performed using the PPP master mix (Top‐Bio) with primers (Generi Biotech) previously described for the detection of C. jejuni and C. coli (Bang et al., 2002 (link); Linton et al., 1997 (link); Winters et al., 1998 (link)). MALDI‐TOF/MS (Autoflex speed TOF/TOF; Bruker) was used for confirmation of C. jejuni and C. coli identification by spectral comparison with the MALDI Biotyper library (MBT 8468; Bruker).