Surecycler 8800 thermal cycler

All fishery samples were refrigerated until they were tested. The surfaces of the cans were cleaned and dried, and the top surfaces were covered with ethanol (96%) and left to stand for 2 min until the ethanol was evaporated. The cans were opened with a sterile can opener and put in large plastic bags to avoid the spread of aerosols. Furthermore, 20 g of each fish was aseptically placed in a sterile mortar with 10 mL of sterile peptone water (0.1%) and then blended for 2 min. Amounts of 2 g of the prepared solid samples were inoculated into two screw-capped bottles; one contained 15 mL of the Trypticase peptone glucose yeast broth (TPGY; purchased from Oxoid Ltd., Basingstoke, Hampshire, UK) while the other contained 15 mL of a cooked meat medium (CMM; obtained from Oxoid Ltd., Basingstoke, Hampshire, UK). The inoculated media were incubated under anaerobic conditions for 7–10 days using an AnaeroGen^TM gas-generating kit (bought from Oxoid Ltd., Basingstoke, Hampshire, UK). The Trypticase peptone glucose yeast broth was incubated at 30 °C, overnight (16 h), to obtain subcultures. Then, the bacterial suspension of this overnight culture that was enriched on the TPGY medium was used for C. botulinum isolation and identification using Multiplex PCR (Agilent Technologies, SureCycler 8800 Thermal Cycler, Santa Clara, CA, USA), as well for DNA extraction.

16S rRNA region was amplified for all the β hemolysin–positive isolates using the primers as described by Dorsch, Ashbolt, Cox, and Goodman (1994), and A. hydrophila ATCC 7966 strain was used as the positive control. Briefly, genomic DNA was extracted from β hemolysin–positive isolates using Bacterial Genomic DNA Purification Kit (HiMedia, Mumbai, India). Quality and quantity of genomic DNA were measured using Nanodrop™ (Thermo Fisher Scientific) and resolved using 0.7% agarose gel electrophoresis. Details of the primers and their product size are provided in Table 1. 16S rRNA gene was amplified using SureCycler 8,800 Thermal Cycler (Agilent Technologies), and the PCR product was eluted using PureLink™ Quick Gel Extraction Kit (Thermo Fisher Scientific). The eluted PCR product was cloned into TA cloning vector pXcmKn12 (Thermo Fisher Scientific) and transformed into Escherichia coli DH5‐α. Transformants were selected on Luria Bertani (LB) agar ampicillin (50 µg/ml) plate by Blue‐white selection method and confirmed by colony PCR. All the clones were sequenced in automated DNA sequencer (Xcelris Labs Limited, Ahmedabad, India).

The process of detecting mtDNA large-scale deletions was conducted following similar protocols used previously, albeit with slight modifications.^{10 (link)} Two divided fragments of the entire mtDNA, which are 7.8 kb and 9.3 kb, were performed by long-range PCR. For the 7.8 kb amplification, 30 cycles were used with 98°C for 10 seconds, 68°C for 30 seconds, and 72°C for 3 minutes 30 seconds, and a final elongation at 72°C for 10 minutes. For the amplification of the 9.3 kb, the cycling conditions used were 30 cycles of 98°C for 10 seconds, 68°C for 30 seconds, and 72°C for 5 minutes, with a final elongation at 72°C for 10 minutes. The reaction mixture used for both amplifications was Phusion High Fidelity (ThermoFisher Scientific, Waltham, Mass, USA) on the SureCycler 8800 Thermal Cycler (Agilent Technologies, Inc., Santa Clara, Calif, USA). The products were then checked on a 1% agarose gel in TAE buffer for 55 minutes at 75 volts.

Genotyping of IFNL4 rs12979860 and rs117648444 (C>T, P70S) was performed using custom TaqMan SNP genotyping assays (Applied Biosystems) on a 7900HT Fast Real‐Time PCR system (Applied Biosystems).
To sequence IFNL4 (from intron 1 to exon 5), we first amplified the gene from genomic DNA by PCR in a reaction volume of 25 μl (200 ng dNTPs and 0.5 U GoTaq DNA Polymerase, Promega) using primers as reported in Table S2 and cycling conditions as described in reference 4. Sequencing was performed as previously reported.⁵²To genotype PDCD1 SNPs, genomic DNA (30‐300 ng) was amplified in a 50 μL reaction mixture containing 10 pmol of each primer (Table S2), 1.25 U Hot Master Taq DNA Polymerase (5 Prime) and 25 μL PreMixJ (MasterAmp PCR, Epicentre, Madison, USA) on a Sure Cycler 8800 thermal cycler (Agilent Technologies). Amplification started with an initial denaturation at 94°C for 3 min, followed by 30 amplification cycles of denaturation at 94°C for 30 s, annealing at 65°C for 30 s, elongation at 72°C for 1 min, and a 10 min final elongation at 72°C. PCR products were subjected to Sanger automated sequencing analysis.

We estimated mtDNA damage using a quantitative ‘long’ PCR-based assay based on the principle that DNA damage slows down or block DNA polymerase advance⁴¹ . This assay has been previously validated in several species (see^{63 (link)}). The levels of lesions were quantified by the amplification of large mitochondrial genomic fragment and normalized by a short mitochondrial fragment (COI gene), which is less likely to be affected by the random damage (see details of design and validation of primers and PCR conditions in Supplementary Methods and Table S5). qPCRs were performed in SureCycler 8800 thermal cycler (Agilent) using Herculase II fusion DNA polymerase (Agilent) and DNA was quantified using PicoGreen (dsDNA assay kit Invitrogen) in a Synergy HT BioTek microplate reader (see details in Supplementary Methods). Relative DNA lesion frequencies were normalized to reference as described by Furda et al.⁶⁴ . Briefly, we estimated the relative damage per DNA strand as the ratio of fluorescence values of large and small mtDNA target in each sample (RS) and in the reference (RR). Normalized mtDNA damage was determined as –ln(RS/RR).

The specificity of each primer pair was checked in preliminary conventional PCR assays with bovine, ovine and caprine cDNA synthesized from Tempus™ total RNA. All PCR reactions were performed with SureStart Taq DNA Polymerase (Agilent Technologies, Inc. Santa Clara, USA) following the manufacturer’s instructions, with 10 ng of cDNA in 0.5 μM of each primer. PCR reactions were conducted on a SureCycler 8800 Thermal Cycler (Agilent Technologies, Santa Clara, USA). The PCR program consisted in an initial denaturation step of 10 min at 95°C, followed by 40 cycles of 30 sec at 95°C, 30 sec at 60°C, and 45 sec at 72°C, followed by a final elongation step of 10 min at 72°C. PCR products were analyzed on 2% agarose gel. Amplicon size was checked with agarose electrophoresis migration (amplicon sizes are listed in Table 2). Finally, PCR products were (i) purified with QIAquick PCR Purification Kit (Qiagen Ltd., Crawley, UK), according to the manufacturer’s instructions, (ii) quantified using a NanoDrop™ ND-1000 Spectrophotometer (Thermo Fisher Scientific, MA, USA), and (iii) sequenced (Beckman Coulter Genomics, Takeley, UK).
The absence of genomic DNA (gDNA) amplification was checked with bovine, ovine and caprine gDNA isolated from blood buffy coat, using the QIAamp DNA Mini Kit (Qiagen Ltd., Crawley, UK).