Portable LAMP Diagnostics for Food Safety

Safe and healthy food is essential for life. With the current shift towards a more sustainable future and the ongoing climate change, new challenges and risks are observed in the food production chain. A sustainable future can only be achieved by complementing systems for food safety and food security. Solutions for increased food security, such as limited food loss and waste, transition towards more plant based food products or recycling food stuffs, pose us for new challenges regarding food safety [1]. Next to the shift towards a sustainable future, a shift in consumer awareness and participation is observed. Consumers expect food on the market to be safe, but at the same time to become more aware of the potential fallibility of the food production system [2]. These new aspects regarding food safety and security, and the increased consumer awareness are, amongst others, the basis for the rise of so-called point-of-need (PON) or point-of-care (POC) tests. POC or PON tests are defined as (medical) diagnostic tools to diagnose quickly and accurately at or near the site where (medical) care is needed [3], meeting key requirements for simplicity, speed, sensitivity, portability, robustness and affordability. Today, such portable testing systems are widely developed and implemented for medical applications, but the need to develop similar systems to assure food safety [4, 5] is urgent. In case of food safety control, a PON or POC test is a portable testing system that enables producers, consumers or inspection authorities to act promptly in case of a non-conformity, such as a pathogen or a fraudulent ingredient in the production process or an allergenic compound in a product not labelled as such. Nucleic acid based diagnostic tools are ideal for point-of-need use due to their high specificity and sensitivity. The tools consist of extraction of the nucleic acids, (isothermal) amplification and specific readout. Challenges for nucleic acid based point-of-need assays to be applied for food safety control lie within the wide range of different matrices and targets, and the possible low abundance of the targets. Next to that, different customers, e.g. producers, consumers or inspection authorities, have different needs regarding the key parameters as speed, cost, multiplexing possibilities and ease of operation. In order to be applicable for food safety control, and to be used at location by producers, consumers or inspection authorities, development of portable and easy-operable nucleic acid based diagnostic tools requires a multifactorial approach. We propose a modular system for quick and simple development of specific nucleic acid based diagnostic tools. The modular system helps customers to assemble a portable system fulfilling their needs. For example, a producer who needs to check the production area for the presence of a pathogen as Salmonella, cost and speed are important key parameters. According to the modular system, the assay can be assembled as follows: surface swabs for sample collection without additional pre-treatment, a simple extraction buffer will suffice as Salmonella is an easy target to extract DNA from and as amplification device the Tcup65 can be used in combination with an LFD read-out in order to have a simple, fast and cost-effective assay. In contrary, retailers who want to test the (raw) materials for possible fraudulent admixtures have different requirements for their portable system. Materials as feed or food products require physical disruption as grinding in combination with a more intrusive extraction buffer in order to breakdown the cell wall, and multiple targets, e.g. different plant species, might be present which makes multiplex analysis a key requirement. The portable system would therefore consist of a more elaborate sample pre-treatment and extraction, in combination with multiplex amplification and real-time readout. Although these examples are concise, they already underline the need for the modular system. All modules will be addressed within this project, but special attention will be given to two topics: LAMP primer design and the incorporation of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease (Cas)-based (CC) applications. The isothermal amplification technique LAMP (Loop-mediated amplification) will be the technique of choice, but although this technique is widely used, successful in silico primer design is more challenging compared to PCR primer design [6]. Performance of LAMP primers can be unpredictable, therefore more knowledge on the characteristics that in silico can determine if a primer set will be successful is beneficial. CC sensing techniques as SHERLOCK [7] and DETECTR [8] are already used as alternative detection method in POC/PON assays [9]. Next to that, Steens et al. [10] described another CRISPR system, able to detect at SNP level. Another CC application is the use of a mutant, inactive form of the Cas protein (dead Cas or dCas) to bind and capture specific targets [11]. This application is especially interesting for low abundance targets, as pathogenic viruses. Incorporation of CRISPR/Cas sensing and/or capturing will increase the robustness, specificity and sensitivity of the assay and will therefore be part of the described modular system. All aspects of the modular system will be addressed within this project, with focus on food-safety related targets. Briefly, the project will be outlined as follows: development of portable assays for food-related targets as Vibrio parahaemolyticus, Salmonella spp., Escherichia coli and norovirus, of CRISPR/Cas-based target enrichment for low abundance targets, of easy-operable devices and development of the modular system. References 1. Vågsholm, I, et al. (2020). Food security, safety, and sustainability—getting the trade-offs right. Frontiers in Sustainable Food Systems, 4, 16. 2. Lord, N., et al. Fault lines of food fraud: key issues in research and policy. Crime Law Soc Change (2021). https://doi.org/10.1007/s10611-021-09983-w 3. Kost, G. J., et al. (2006). Point‐of‐Care Testing: Principles, Practice, and Critical‐Emergency‐Disaster Medicine. Encyclopedia of Analytical Chemistry: Applications, Theory and Instrumentation. 4. Choi, J. R., et al. (2019). Emerging point-of-care technologies for food safety analysis. Sensors, 19(4), 817. 5. Campbell, V. R., et al. (2021). Point-of-Need Diagnostics for Foodborne Pathogen Screening. SLAS TECHNOLOGY: Translating Life Sciences Innovation, 26(1), 55-79. 6. Meagher, R. J. et al. (2018). Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA. Analyst, 143(8), 1924-1933. 7. Gootenberg, J. S., et al. (2017). Nucleic acid detection with CRISPR-Cas13a/C2c2. Science, 356(6336), 438-442. 8. Chen, J. S., et al. (2018). CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science, 360(6387), 436-439. 9. Van Dongen, J. E., et al. (2020). Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities. Biosensors and bioelectronics, 166, 112445. 10. Steens, J. A., et al. (2021). SCOPE enables type III CRISPR-Cas diagnostics using flexible targeting and stringent CARF ribonuclease activation. Nature communications, 12(1), 1-12. 11. Slesarev, A., et al. (2019). CRISPR/Cas9 targeted CAPTURE of mammalian genomic regions for characterization by NGS. Scientific reports, 9(1), 1-12.