Biometra tone thermocycler

The Class I, Class II, and Class III integrons were detected based on the presence of gene sequences characteristic for Integrase 1 (IntI1), Integrase 2 (IntI2), and Integrase 3 (IntI3), respectively. The presence of genes encoding the integrases Int1, Int2, and Int3 was evaluated by a polymerase chain reaction (PCR) in all isolates (n = 193) using the primers and conditions previously described (Supplementary Table S1) [34 (link),35 (link)].
DNAs from bacterial strains from INIAV and the European Union Reference Laboratory for Antimicrobial Resistance (DTU Food, National Food Institute, Denmark) were used as positive controls in all PCR amplifications. The amplification reactions were run in a Biometra TOne thermocycler (Analytik Jena, Jena, Germany).
PCR products were analyzed by electrophoresis on agarose gel in Tris-Borate-EDTA (TBE) buffer. The products were then visualized and photographed on an ultraviolet transilluminator (BioDoc-It Imaging System, BioRad, Hercules, CA, USA).

Total DNA was extracted from a 4-day aerobic culture (OD at 600 nm of 0.97) in 200 mL R2A medium incubated at 17°C, using the MasterPure complete DNA and RNA purification kit as described by the manufacturer (Lucigen, WI, USA). The 18S rRNA gene was PCR-amplified from total DNA (25 ng) using primers Dios20F (5′-GTGCGTCTGATTCTTGACTCC-3′) and Dios11R (5′-CCCGACCGTCCCTATTAATCA-3′) and DreamTaq DNA polymerase, as recommended by the manufacturer (Thermo Fisher Scientific Baltics, Vilnius, Lithuania). The PCR program (Biometra TOne thermocycler, Analytik Jena, Jena, Germany) involved DNA denaturation at 95°C for 5 min, 30 cycles of 45 s at 93°C, 20 s at 56°C and 1 min at 72°C, and a final 10 min extension at 72°C. The amplified 1,080 bp PCR fragment was sequenced by the Sanger method (Microsynth France, Vaulx-en-Velin, France).

Total RNA from HNEpC was extracted using the RNeasy^® micro kit from Qiagen (Hilden, Germany). RNA extracted from nasopharyngeal samples and HNEpC were reverse-transcribed with the SuperScript™ First-Strand II Synthesis System (Invitrogen, Waltham, MA, USA) using random hexamers (Themo Fisher Scientific, Carlsbad, CA, USA) on the Biometra TOne Thermocycler (Analytik Jena AG, Jena, Germany). The quantification of IFNA, IFNB, IFNL1, IFNL2/L3, IFITM1, IFITM3, MX1, DEFB1, DEFB4A and DEFB103 mRNA by qPCR was performed on a LC480 (Roche Diagnostic) using SYBR^® Green I Master mix (Thermo Fischer Scientific). Relative quantification was performed using the method developed by Vandesompele et al. (69 (link)), using RPS18 and EF1A as references. The calculation method is based on the conversion of the linear Cq values into a logarithmic scale using the efficiency of PCR as an exponential function. The geometric mean of selected housekeeping genes is used as a normalization factor, allowing eliminating inter-sample variations. The value 1 is given to the calibrator with the highest expression level within a series; the levels of expression of the other genes are then calculated compared to the calibrator. Primer sequences are listed in the Supplementary Table S1.