Fast and Sustainable Thermo-osmotic DNA Extraction Protocol for Trans-spectrum Contingency and Field Use

Background

A fast and massive DNA extraction is quintessential for nucleic acid amplification tests (NAAT), especially if performed under duress; the latter implies either field conditions in resource-limited settings (RLS), in routine or expedient setups, or in abnormal conditions with samples surging and/or supply pipelines malfunctioning due to corporate concerns or supply chain overextension/collapse. Thus, although the technology for DNA extraction systems achieves significant yields from complicated and/or miniscule samples, their use is expensive even if purchased in bulk to achieve economies of scale. Alkaline approaches were suggested for contingency (Goudoudaki et al., 2021) and field conditions (Priye et al., 2016), adaptable to the intended use (Goudoudaki et al., 2021). Still, specific chemicals were used, which may not be available in proper quantities once a health or ecological crisis prompts massive spatial dispersion and increased processing output of incoming samples, which may overload standard public health or agronomic facilities and infrastructures. Thus, ease of procurement and use by personnel moderately trained in this specific discipline but experienced in other sectors of biosciences may increase the processing rate of initial screening and offload work from dedicated infrastructures, restricting their involvement to tackle only unresolved or suspect samples. Last but not least, this almost in-situ processing approach may limit the need for dispatching samples to distant facilities, which is expensive, time consuming, creates biosafety and biosecurity risks, and may degrade the samples, thus de-valuing the actual diagnostic procedure.

By using only distilled water and heat, both easily available, there is no chemical waste and no need for special reagents, and thus for diverse stockpiles. The latter may be inexpensive and long-lead or long-expiration date, but their storage, even in normal conditions, takes space and there is a need for correct management of the stock. Also, the skills necessary are basic, found in everyone with the most elementary training in Biosciences. Thus, the overall footprint of the analysis, both before- and after-use, is kept minimal. This protocol is actually an expansion of a previous one, developed during the COVID-19 pandemic (Fomsgaard and Rosenstierne, 2020) as a result of kits supply restrictions (Benda et al., 2021), to include different extraction substrates and target genomes. It can be used in the field, given that consumables, heat, and electricity sources for portable thermocyclers (Kambouris et al., 2020) can be provided. Compared to the alkaline protocol (Goudoudaki et al., 2021), the thermo-osmotic protocol described herein requires even less reagents and produces no chemical waste, nor does it require stocking reagents that may expire and require specific storage. The protocol is named after the thermal deterioration of the membrane bonds allowing osmosis to perform the cell lysis and has been used already in one original publication (Goudoudaki et al., 2023).

Additionally, the present protocol may be used for improving the turnaround time and volume throughput in existing, indoor facilities. This comes at the expense of other performance metrics including sensitivity, which are markedly inferior compared to the ones achieved by dedicated DNA extraction protocols, even those of relatively low cost (Velegraki et al., 1999a and 1999b). Furthermore, facilities commandeered during crisis management may be among the beneficiaries. The method may be used for public health and environmental and agronomic emergencies (Kambouris et al., 2018), thus qualifying for compatibility with the One-Health framework and any other that may refer to genetic/genomic biomarkers that may be typed by basic PCR protocols, with or without follow ups such as, but not restricted to, restriction digestion (Velegraki et al., 1999a and 1999b; Arabatzis et al., 2004) and single-locus sequencing (Irinyi et al., 2015). The method, being plain and simple, shows moderate performance and should be used for highly enriched (containing high numbers of target genomes) and uncomplicated (without similar loads of pseudo-targets and inhibitors) samples.