Gspssd isothermal mastermix iso 001

LAMP reactions were carried out as suggested in the OptiGene Ltd. LAMP User Guides (Version 1.1, OptiGene Ltd., Horsham, UK). For each LAMP reaction, a 25-µL reaction mixture containing 15 μL of GspSSD isothermal mastermix (ISO-001) (OptiGene Ltd.), 2.5 μL primer mix, 2.5 μL PCR-grade water (Qiagen GmbH, Hilden, Germany), as well as 5 μL template, was prepared. LAMP reactions were performed using the real-time fluorometer Genie^® II (OptiGene Ltd., Horsham, UK).

The newly designed LAMP primers amplified a 254 bp DNA fragment of the COI gene. All qLAMP reactions were performed with a sample volume of 2 µL to achieve a final volume of 20 µL. For each reaction, 10 µL of GspSSD Isothermal Master Mix ISO-001 (OptiGene Ltd., Horsham, UK) were used. The reaction was optimized attending to primers concentration, amplification temperature and reaction supplementation with Betaine and dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Darmstadt, Germany). The amplification temperature was evaluated in the 60 °C to 65 °C range for optimization. Regarding reaction supplements, Betaine was tested at 0.6 M, 0.8 M and 1 M, while DMSO was tested at 5% and 7.5%.
The concentration of primers used was 1600 nM for FIP/BIP, 200 nM for F3/B3 and 800 nM for LB, being DNA amplification of the environmental samples performed at 64 °C for 1 h without supplements. In addition, a melt curve step was included in all qLAMP experiments, consisting of heating up to 98 °C for 15 s, cooling down to 75 °C for 1 min and heating up to 95 °C for 15 s, acquiring fluorescence every 0.3 °C.

In accordance with the manufacturer’s instructions, the LAMP assay based on gene gyrA was carried out with the five T. bernardiae isolates, type strain T. bernardiae DSM 9152 ^T, and with control strains of genus Trueperella and closely related genus Arcanobacterium. A total volume of 25 µL for each reaction included 15 µL GspSSD isothermal master mix (ISO-001) (OptiGene Ltd., Horsham, UK) and 2.5 µL primer mix (ISO-001; OptiGene Ltd.), gyrA- F3 primer, and gyrA-B3 primer with a final concentration equivalent to 0.2 µmol/L, gyrA-FIP primer, and gyrA-BIP primer with final concentration equivalent to 0.8 µmol/L and gyrA-LoopF Primer and gyrA-LoopB Primer with a final concentration equivalent to 0.4 µmol/L. Subsequently, 5 µL DNA was added as a template. The LAMP assay was run at 70 °C for 20 min with a melting curve analysis step (annealing curve 98 to 80 °C ramping at 0.05 °C/s) in a real-time fluorometer GenieII® (OptiGene Ltd.).