Iso 001

All LAMP reactions were conducted in the B-cube device (Hyris, London, UK) in 16- well cartridges. Each reaction was 25 µL final volume comprising 15 µL of the isothermal master mix ISO-004 or ISO-001 (OptiGene, Horsham, UK), which vary in the MgSO₄ concentration, and 5 µL of the template DNA. In this step only DNA from G. pallida was used. For all primers (Table 1), five different concentrations were tested in different combinations during the optimization process. For the FIP and BIP primers (50 µM) the volume varied from 0.4 to 0.9 µL and was combined with different volumes of the F3 and B3 outer primers (50 µM), which varied from 0.10 to 0.15 µL each. For the optimization of the temperature and time, the reaction mixtures were incubated at 65 to 63°C, for 60 to 20 min. To determine the product melting temperature, the generated products were heated from 75 to 95 °C at a rate of 0.05 °C·s⁻¹. In all LAMP assays, as a negative amplification controls (NAC), 5 µL of water was added to the reaction instead of DNA extract. The LAMP products were detected by the SybrGreen fluorescence.
The protocols in Table 3 were tested during the optimization of the LAMP protocol for G. pallida identification.
Briefly, at the end, the LAMP reactions should be performed as described in Table 4.

rRT-LAMP was performed using either Dukes et al. (2006) (link) primers in both wet (rRT-LAMP-d-wet) and lyophilised (rRT-LAMP-d-dry) formats as previously described (Howson et al., 2015 ), or using primers from Toshiba Medical Systems Corporation [TMSC] (rRT-LAMP-T-wet). rRT-LAMP-T-wet was performed in a total reaction mixture of 25 μl, containing: 15 μl isothermal mastermix ISO-001 (OptiGene Ltd, Horsham, UK.), 2.5 μl TMSC FMDV primer mix (a proprietary RT-LAMP primer mix targeting the 3D^pol-coding region), 0.15 U Avian Myeloblastosis Virus (AMV) reverse transcriptase (RT) (OptiGene Ltd.), 5 μl sample and made up to total volume with NFW. For all rRT-LAMP formats, reactions were performed in duplicate as previously described (Howson et al., 2015 ) using a Genie^® II (OptiGene Ltd.), with rRT-LAMP-T-wet incubated at 63 °C for 30 min. Time to positivity (T_P) and anneal temperature (T_a) calculations were automated using Genie^® Explorer v0.2.1.1 software (OptiGene Ltd.). Samples were called positive if amplification had occurred and the rRT-LAMP product annealed in the amplicon-specific temperature range (87.5–89.5 °C for Dukes et al. (2006) primers (Howson et al., 2015 ), 87.7-89.7 °C for TMSC primers)(Table 1).