Isolate 2 pcr and gel kit

For DNA sequencing, the amplification products obtained from the PCR reactions were purified with the ISOLATE II PCR and Gel Kit (Bioline, London, UK) and sequenced in an Avant 3100 sequencer (Applied Biosystem, Waltham, MA, USA). Both strands used the same set of internal primers as in respective PCR assays. DNA sequencing was performed by a sequencing facility at the University of Veterinary Medicine, Košice, Slovakia. The obtained DNA sequences in the partial bg and tpi genes were compared with the reference sequences of G. duodenalis in the GenBank database by the nucleotide BLASTn program [15 ]. Sequences were sorted by similarity and aligned using the Clustal Omega tool [16 (link)]. Phylograms were constructed by the MEGA7 software [17 (link)] using the maximum-likelihood (ML) method with 1000 bootstrap pseudoreplicates. A TN93 substitution model with gamma-distributed rate heterogeneity [18 (link)] was selected based on the maximum likelihood evaluation of possible substitution models using the Modeltest in MEGA.
The gene sequences for 13 isolates examined in tpi were deposited in GenBank^® under accession numbers OL456145-OL456151 and OL474006-OL474011, and under accession number OL581604 for isolate examined in bg.

A genome sequence spanning the V3 and V4 regions of the 16S rRNA gene were amplified using the Illumina ultramer oligonucleotides (Integrated DNA Technologies, Coralville, IA) that include Illumina adapter overhang nucleotide sequences (50 (link)). Polymerase chain reaction (PCR) conditions and reagent volumes are available as Supplementary Information. A negative control from each gDNA isolation step was included in amplification steps to determine the possibility of carry-over contamination between samples and reagents during these steps. Amplification products were purified using the ISOLATE II PCR and Gel Kit (Bioline) according to the manufacturer's instructions with minor adjustments (see Supplementary material).

For genotyping, genomic DNA was extracted from freeze-dried 2-week-old plant material using a phenol–chloroform method as described by Rogowsky et al. (1991) (link). PCR was performed using Q5® high-fidelity DNA polymerase (New England BioLabs, USA), following the manufacturer’s protocol in a final volume of 12.5 μl. All PCR products were separated by agarose gel electrophoresis, purified using the ISOLATE II PCR and Gel Kit (Bioline, Australia), and Sanger sequenced at the Australian Genome Resource Facility (AGRF). Primers used for PCR can be found in Supplementary Tables S3–S5.

For a precise evaluation of PCR sensitivity, we constructed a plasmid containing an insert of the target sequence amplified from the Ppa F149 strain. For this purpose, the product of PCR amplification was purified using ISOLATE II PCR and Gel Kit (Bioline, St. Petersburg, Russia) and cloned to pAL2-T vector using a QuickTA kit (Evrogen). Plasmid DNA used as standard was purified with a QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Sanger sequencing of the corresponding region in the resulting plasmid confirmed the correctness of the insert.

PCR was performed with designed primers: forward 5′-CTCCCTCCCTCCCCGATTAC and reverse 5′-CCGGACACCTGGAAGAACCC. The reaction mix contained 1 × Taq-buffer (2.5 mM Mg2+); 200 μM dNTP; 2,5 U Tag DNA polymerase (Sileks, Moscow); 0.4 μM of each primer; ~ 30 ng of template DNA, 5% DMSO and sterile distilled water in a final volume of 25 μl. PCR cycling conditions consisted of an initial denaturation step of 95 °C for 3 min, followed by 30 cycles of 95 °C for 20 s, 63 °C for 30 s, 72 °C for 45 s, and a final extension cycle at 72 °C for 5 min. PCR products were detected on 1,3% agarose gel in TBE buffer, then were purified with ISOLATE II PCR and Gel Kit (Bioline) and sequenced in two ends (Eurogen, Moscow) using forward and reverse primers. The DNA sequences were aligned with Unipro UGENE software [13 (link)]. GenBank sequence accessions are: ‘Barke’ KX611232, ‘Triumph’ KX611233, ‘Morex’ KX611234, ‘Franklin’ KX789375.
To reveal the 7 bp deletion in exon 1 of HvGA20ox2 gene 3 μl of the PCR product were digested with 1 U of Hinf I (Sibenzyme, Novosibirsk) in a total reaction volume of 15 μl containing 1х SEbuffer O (pH 7.6) for 2 h at 37 °C, followed by electrophoretic separation in 1.5% agarose gel in TBE buffer.

A dsDNA gBlock Gene Fragment (Integrated DNA Technologies Inc., Coralville, IA, USA) was synthesised as a positive control for ASFV and initial primer optimization. The gBlock was made by aligning the two outer LAMP primers (F3/B3) [36 (link)] (Table 1) to Malawi Lil-20/1 ASFV genome (GenBank accession AY261361), covering a 206 bp region of the TPII (Table 2). The gBlock was amplified using 1 µL of each F3 and B3 ASFV LAMP primer at 10 µM using Platinum^TM PCR SuperMix High Fidelity (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. The amplified product was purified using an Isolate II PCR and Gel Kit (Bioline, Taunton, MA, USA) and quantified using dsDNA HS Assay Kit on a Qubit^TM 2.0 (Invitrogen Carlsbad, CA, USA) fluorometer. A second gBlock was designed to be used as an exogenous internal amplification control (IAC). Briefly, the IAC gBlock targeted the same region as the ASFV gBlock apart from the addition of 8 nucleotides (Table 2) to elevate the annealing temperature. The IAC gBlock was prepared as outlined above.

DNA samples from ear-tag and ovarian tissue were extracted using the Isolate II Genomic Kit (Bioline, London, UK), according to the manufacturer’s protocol. To 200 ng of genomic DNA, 5 μL of 10X NH₄ Reaction buffer, 1.5 μL of 50 mM MgCl₂ solution, 0.5 μL of 50 mM dNTPs, 1 μL of 25 μM of each forward and reverse primer (F: 5′-GCATTCCATTCGTATGCAAACC-3′; 5′-ATTGTCGTGCCGGATCATGA-3′), 2.5 U of BioTaq Polymerase™ (Bioline) and 38.5 μL of ddH₂0 ultrapure water were added. Samples were amplified on a thermocycler (MJ Research DNA Engine PTC 200) at 95 °C for 5 min, and 34 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s with a final elongation step of 72 °C for 5 min. Products were separated on 2 % agarose gels at 100 V for 60 min, and visualised under UV. Products were purified using the Isolate II PCR and Gel Kit (Bioline), according to the manufacturer’s protocol.