## 3 Results and Discussion
### Long-Term Stability at Room Temperature  
To investigate the stability of the LAMP reaction mixtures over time, the glycerol-free Bst 3.0 polymerase (spin-filtered) and the GFP were used. The reaction mixtures were prepared with two different primer sets, lyophilized and stored for up to 24 weeks (168 days) before real-time measurements were performed. The results using the plaie-specific primer set are shown in Figure 1A and the results of the safflower-specific primer set in Figure 1B. Both assays demonstrated that the LAMP reaction can be carried out with both polymerases even close to 6 months of storage time at room temperature if the reaction mixtures have been suitably dried (freeze-drying with 0.3 M trehalose as stabilizer). For the safflower-specific primer set, the spin-filtered Bst polymerase showed an increase in reaction time (hereon: time to positive, tp) of 10 min after seven days of storage compared to the first day of storage. A further increase of 10 min in reaction time was observed for storage up to 168 days. No significant differences in reaction times were observed for the different storage days compared to the mean reaction time over all storage days. Moreover, no significant difference was observed between the first and the following days of storage. For the plaice-specific primer set reaction, a relatively high variability in assay ttp was observed over the period of 150 days, although there was no actual loss of activity of the freeze-dried reaction mix. The ttp after a seven-day storage at room temperature was an average of 31.29 \(\pm\) 0.99 min compared to 26.80 \(\pm\) 1.48 min after 150 days. The reaction for 28 and 84 days of storage failed to generate results. Due to the lack of stored reaction mixtures, it was not possible to repeat the measurements. Although significant differences between individual storage days were observed, due to the high variability of the assay ttp, statistical tests revealed no significant differences between the individual storage days and the mean reaction time over all storage days. The GFP also showed no significant differences between the individual storage days and the mean reaction time over all storage days. Furthermore, the extension of the reaction time after storage for the safflower-specific primer set for 7 days (16.93 min) and 112 days (14.46 min) revealed no significant difference. The plaice-specific primer set reaction showed relatively constant reaction times (statistically no significant differences) in the first 54 days after lyophilization (7 days: 32.71 \(\pm\) 1.18 min; 28 days: 28.73 \(\pm\) 0.94 min; 54 days: 31.31 \(\pm\) 1.03 min). However, after 72 days, the reaction time increased dramatically to 42.80 \(\pm\) 1.18 min. The ttp after 72 days of storage was significantly different compared to the mean reaction time over all storage days. In order to determine whether the ttp on day 72 was an outlier, further storage days would have to be tested. The conducted tests were stored at room temperature for the experiments. Hence, observing similar reaction dynamics after two months for both assays is a promising result to ensure robustness for REASSURED testing. In contrast to our results, [PERSON] et al. did observe a significant increase in the time-to-positive rate after only 12 days of storage of LAMP reaction mixes at room temperature, even though trehalose and glycine were used as stabilizers. Therefore, they recommend storage of lyophilized reaction mixtures at 4 \({}^{\circ}\)C. It was not discussed by the authors if glycerol in the storage buffer might have led to loss of activity. Moreover, a lyophilized RT-PCR assay specific for _Myobacterium tuberculosis_ stored at room temperature showed poor performance already after two weeks, despite the different cryoprotectants that were used. Only when stored at \(-\)20 \({}^{\circ}\)C and 4 \({}^{\circ}\)C did the assay show a shelf life of up to 6 months [35]. Storage temperature has a significant impact on shelf life.