REASSURED Test System for Food Control--Preparation of LAMP Reaction Mixtures for In-Field Identification of Plant and Animal Species  
[PERSON]  
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analytical laboratories. As shipping suspicious samples to analytical laboratories is time-consuming and costly, detection systems that can also be used on site generally represent significant benefits for users. In 2003, the World Health Organization (WHO) published a set of criteria for ideal point of care test systems. These criteria are known by the acronym ASSURED (affordable, **s**ensitive, **s**pecific, **u**ser-friendly, **r**apid, **e**quipment-free, **d**elivered). Recently, ASSURED criteria were updated to provide real-time quality control (R) and the ease of specimen collection (E), building REASSURED criteria [5; 6]. REASSURED criteria not only can be used for development of medical POC test systems, but also represent a promising basis for the development of test systems for the surveillance of food commodities. Developed tests should be easy to perform in 2-3 steps (sampling, extraction and specific amplification coupled with intuitive read out) and should only require minimal user training with no prior knowledge of diagnostic testing. For the amplification of target DNA at the POC, the reduction in necessary equipment is especially relevant. Since its publication by [PERSON] et al. in 2000, the LAMP (loop-mediated isothermal amplification) reaction has become one of the most widely used isothermal DNA-amplification methods [7]. Due to its isothermal nature, the reaction is particularly well suited for DNA analysis in resource-limited environments [8]. The advantage of LAMP is the high sensitivity and sequence specificity, although off-target effects, such as unspecific amplification or cross-reactivity, have been described [9]. Several LAMP assays have been published for use in food analysis [10; 11; 12]. In general, publications on the topic of LAMP reactions often emphasize its potential for on-site analysis due to its isothermal nature, robustness against inhibitors and the variety of visual detection methods [13]. However, the problems that can arise during on-site analysis are usually scarcely addressed. These include, in particular, the transportation and storage of the required reagents (i) and the intuitive visualization of the assays result (ii). The LAMP assays still require fresh preparation of the LAMP reaction mixtures to perform, and therefore fail to fulfill the REASSURED criteria, especially the robustness, equipment-free and delivered criteria, completely [14]. To our knowledge, only two publications on the detection of different species in food showed the performance of DNA isolation and the LAMP reaction at the POC [15; 16]. However, multiple heating steps and laborious temperature control prevent user-friendly operation [16]. Ensuring an uninterrupted cooling chain when transporting a ready-to-use LAMP reaction mix to the POC is mandatory and involves considerable costs. Storing LAMP reaction mixes (i) in lyophilized form not only overcomes instability issues but also enhances the user-friendliness of the tests and therefore affects the REASSURED criteria in more than one way. Various enzymes, including Bst polymerase, retain their activity in freeze-dried form after reconstitution [17; 18; 19; 20]. During the process of lyophilization, the protein is exposed to drying and temperature stress, resulting in the risk of (partial) denaturation of the protein and subsequent loss of enzyme activity. To protect the enzyme during the drying process, protein stabilizers (lyoprotectants and cryoprotectants) must be added. These are, for example, saccharides that interact directly with the proteins and form a protective layer on the protein surface [18]. The disaccharides sucrose and trehalose are frequently used as stabilizers.