## 3 Results and Discussion
### Reproducibility  
The reaction time of the LAMP reactions varies between the individual measurements, although the reaction mixtures were prepared at the same time. It was therefore necessary to check whether the fluctuations were due to storage or to individual effects during lyophilization (Figure 2). In order to verify the cause of the fluctuations, 20 individual reaction mixtures were prepared for each primer set and each polymerase, lyophilized and measured after seven days of storage. Reaction tubes with a volume of 0.2 mL were used for this purpose. Instead of the previously used Beta 2-8 LSCplus freeze-dryer, the smaller Alpha 2-4 LSCplus freeze-dryer was used, which enabled better performance for small quantities and small volumes. All prepared single tubes were stored at an ambient temperature (20 \({}^{\circ}\)C \(\pm\) 2 \({}^{\circ}\)C) in a desiccator under exclusion of light for seven days. After one week, all reaction tubes were resuspended with 24 \(\mu\)L of ultrapure water. A total of 15 reactions were performed in replicates with the target DNA (1 ng or 10 ng) and 5 reactions were performed in replicates with non-target DNA.  
As shown in Figure 2, both the GFP and the spin-filtered Bst 3.0 had a low standard deviation. For the lyophilized reaction mix specific for _Pleuroncetes platessa_ (_P. platessa_), the average time to positive was 15 min \(\pm\) 0.82 min for GFP and 17.69 min \(\pm\) 0.99 min for Bst 3.0. For the lyophilized reaction mix specific for _Carthamus titorius_ (_C. titorius_), the average time to positive was 11.43 min \(\pm\) 2.10 min for GFP and 12.12 min \(\pm\) 1.52 min for Bst 3.0. Both assays showed low variation in amplification time and, thus, enabled robust POC testing with no refrigerated storage needed. Interestingly, both assays revealed overall faster times to positive when compared to the long-term stability tests described above. This is likely due to the variable performance of the device used for lyophilization.  
The LAMP technique is known to produce reproducible results over a wide range of temperatures, elongation times and pH values. However, as described above, most of the published research has used freshly prepared reaction mixtures. For user-friendly POC application, cooling the reaction mix prevents ease of use. It has been demonstrated that lyophilized reaction mixtures can be stored for up to 24 weeks. The reproducibility of single lyophilized tubes is, to the best of our knowledge, only described in detail for colorimetric LAMP assays. [PERSON] et al. (2022) designed a test kit for SARS-CoV-2 RNA based on lyophilized colorimetric RT-LAMP. Their test kit remained stable for at least 30 days at 4 \({}^{\circ}\)C and 10 days at room temperature (20-22 \({}^{\circ}\)C). [PERSON] et al. achieved high sensitivity and a >99% specificity with a reproducible LoD of 100 RNA copies per reaction under both storage conditions [37]. Recently published results of [PERSON] et al. (2024) support the fact that lyophilized colorimetric RT-LAMP assays show high stability at room temperature for up to 6 weeks. The authors described those standard thermal reactions with SARS-CoV-2-positive RNA as showing a functional and stable reaction [38].