Scalable Alkaline Extraction Protocol for Microbial DNA Screening by PCR

Background

In routine conditions or in crisis, surveillance and screening require fast and massive DNA extraction pipelines for nucleic acid amplification tests (NAAT), especially PCR, both in field conditions [usually in resource-limited settings (RLS)] and in proper lab setups. Although current technology for DNA extraction achieves significant yields from complex and/or small samples, their use is expensive and subject to market manipulation and practices and supply chain integrity [1,2]. The alkaline approach has been suggested for contingency [3] and field conditions [4]. It is a straightforward and inexpensive method, less susceptible to supply chain and market fluctuations as it uses only widely available, inexpensive chemicals and expendables. In particular, NaOH—the main lysis factor—is much more efficient than ultra-short, detergent-based approaches, either as kits or as household formulations [5] or boiling water [6]. Due to its scalability, which is its novelty, the protocol described herein may result in a template for immediate use in NAAT (mainly PCR) or in an extract that is storable, transportable, much more enriched, and amplification-friendly. In both cases, the serial dilutions just before use overcome the lack of a spectrophotometer in RLS to manage concentration uncertainties [7].

In a health or ecological crisis, massive spatial dispersion and increased processing output of incoming samples may overload and overrun standard public health or agronomic facilities and infrastructures. Thus, ease of procurement and use by personnel moderately trained in molecular diagnosis but experienced in other sectors of biosciences may increase the processing rate in point-of-collection (PoC) settings, thus neutralizing infectious potential and offloading work from dedicated infrastructures; the latter would tackle unresolved or suspect samples and provide confirmatory analyses as needed. The choice for in situ refinement allows safe dispatching of samples to distant facilities [8], which is otherwise expensive, creates biosafety and biosecurity risks, and may degrade the samples, thus devaluing the actual diagnostic procedure.

The present protocol may be used in RLS, ideally combined with portable instrumentation [9] in its crude, fast version for immediate testing in time-sensitive scenarios, and/or severe RLS. In more permissive scenarios and conditions, its refined version may be implemented, producing much better results [3]. This protocol may be used to improve the turnaround time and volume throughput in established, indoor facilities, especially in its refined version, to increase sensitivity. This comes at the expense of other performance metrics, including speed and simplicity. This method may be used for public health and environmental and agronomic emergencies [10], thus qualifying for implementing the One-Health framework. Processing uncomplicated human/animal samples for genotyping genomic biomarkers by basic PCR protocols or other NAAT should be relatively straightforward and requires no modification, as such samples are easier than the ones tested in the validation publication. Blood is always an unknown quantity regarding extraction efficiency and robustness; less demanding samples, such as mouth swabs, are easy to process and sufficient for targeted biomarker analyses since they are within the capability limits of the less robust thermo-osmotic protocol [6]. Additionally, simplex, single-locus PCR results may be further analyzed downstream by restriction digestion [11,12], single-locus sequencing [13], or any other approach.

Prospective operators should be aware of some limitations of this protocol. Namely, the extract is impure, and thus quantification by standard methods is inapplicable. For the same reason, DNA may be degraded due to a number of factors directly affecting its duration (and thus some of them alleviated in the refined version); the same stands for inhibitor molecules that may hamper the NAAT step. The use of different mechanical rupture techniques, such as sonication, is always a possibility but is not included in this protocol. Enzymatic cell lysis may also be tried; however, it is possible that the chemical environment, due to the NaOH, may degrade or nullify the enzymes used, and thus extensive modification may be required. In any case, the use of enzymes clearly undermines the lean, minimal footprint, supply-chain resilient, and affordable nature of this protocol as presented herein and has not been tested.