Although vaccines have helped alleviate the negative impact of COVID-19, its persistence across many areas of the globe reveals a continuing need for rapid, accurate, and economical COVID-19 testing. But is it even possible to isolate and detect viral RNA rapidly and economically? Here we highlight a repurposed tool for improved SARS-CoV-2 detection: Chelex 100 Resin and the related InstaGene Matrix for rapid, cost-effective viral RNA preservation and detection.
Numerous assays have been developed for SARS-CoV-2 detection, including rapid antigen tests, but the gold standard method for sensitive SARS-CoV-2 detection remains RNA amplification by reverse transcription quantitative PCR (RT-qPCR) (Mardian et al. 2021). However, limited availability and cost of RNA extraction kits can bottleneck the broad implementation of this critical testing method, as we saw early in the pandemic.
Current RNA extraction kits are based on either spin/vacuum columns or paramagnetic beads. Typical column- and paramagnetic bead–based methods require multistep processes that are slow, low throughput, and require various consumables. Additionally, they can lead to operator fatigue when large numbers of samples are processed manually. Automated solutions can address some of these pain points, but in resource-limited settings the additional equipment cost can create further challenges. Therefore, since the onset of the pandemic, there have been intense efforts to find methods that bypass the need for commercial RNA extraction kits which are costly and limited in supply.
One such alternative, which has been increasingly recognized by the scientific research community for the benefits of low cost and speed is Chelex 100 Resin. Chelex 100 Resin and related InstaGene Matrix (research use products) have a history of use for molecular biology applications, but with the supply chain challenges posed by the pandemic they are finding a new niche: preparation of RNA.
Simple, Rapid, and Cost-Effective RNA Preparation
Chelex 100 Resin is a heat-stable bead polymer that can effectively capture divalent cations, such as Mg2+or Ca2+ from solutions. This property can be exploited to inhibit nucleases that require a divalent cation as a cofactor, thus preserving DNA or RNA molecules for downstream detection. In fact, Chelex 100 Resin forms the base of InstaGene Matrix, a ready-to-use suspension originally formulated for DNA preparation.
Traditionally, Chelex 100 Resin–based methods are referred to as ‘extractions’. While a reduction in sample complexity can be achieved using Chelex 100, it is actually intended for nucleic acid preservation, not isolation, and eliminates lengthy nucleic acid purification procedures. Because of this property, Chelex 100 Resin–based methods are sometimes described as “extraction-free.”
The exact method of nucleic acid preparation using Chelex 100 Resin varies by sample type, but a typical procedure offers simplicity and speed: add Chelex 100 Resin bead suspension to a sample, mix and heat to lyse cells and denature macromolecules, and separate beads from the remaining sample by centrifugation (or allow beads to settle); the resulting supernatant contains the nucleic acids. With this basic procedure, nucleic acids can be prepared from a sample in as little as 2 to 5 minutes. Furthermore, Chelex 100 Resin–based preparations do not require additional reagents, except when indicated for specific sample types and downstream applications, making it extremely resource-effective.
A major benefit of the Chelex 100 Resin-based methods is its cost-effectiveness. Researchers recently reported (Guan et al. 2021) an estimated 8-fold cost reduction for SARS-CoV-2 detection when using Chelex 100 Resin. Another study (Lienhard and Schäffer 2021) stated that sample preparation using Chelex 100 Resin was ~170-fold cheaper than using a traditional DNA extraction kit.
The RNA Extraction Kit Dilemma: Solved
Numerous research groups have assessed the performance of Chelex 100 Resin for SARS-CoV-2 detection. Recently, Guan et al. 2021 compared sample preparations using Chelex 100 Resin to commercially available RNA extraction kits. Chelex 100 Resin effectively preserved SARS-CoV-2 RNA in the samples, permitting direct detection by both RT-qPCR and RT-Droplet Digital PCR (RT-ddPCR) and therefore obviating the need for RNA extraction kits. Similarly, another research group (Ulloa et al. 2020) eliminated the use of RNA extraction kits, without compromising sensitivity or specificity, by combining Chelex 100 Resin with a simple nucleic acid precipitation step that uses inexpensive reagents.
Broad Applicability for Molecular Detection
Chelex 100 Resin–based methods are not just suitable for RT-qPCR and RT-ddPCR; they can also be used for reverse transcription loop-mediated isothermal amplification (RT-LAMP), a frequently discussed method for surveillance testing. RT-LAMP is not as complicated to perform as RT-qPCR or RT-ddPCR, requiring only a single temperature for amplification, and can be run on a standard PCR machine or heating block. While its sensitivity may be lower than that of RT-qPCR or RT-ddPCR, RT-LAMP generally yields results much more quickly and with reduced equipment requirements. And indeed, several recent studies (Howson et al. 2021, Yaren et al. 2021, and preprint: Flynn et al. 2020) demonstrate the suitability of Chelex 100 Resin-based methods and RT-LAMP for COVID-19 surveillance.
Safety and Other Benefits of Chelex 100 Resin–Based Methods
Not only do Chelex 100 Resin and InstaGene Matrix reduce costs and sample processing times, they can also enable safer workflows. This is because the heating step that is often part of these sample preparation methods can inactivate viruses such as SARS-CoV-2 even before the sample collection tube is opened, limiting exposure of laboratory personnel to live virus. Guan et al. 2021 demonstrated this benefit when they showed that not only can samples be collected and prepared in a single tube containing Chelex 100 Resin, but that this method actually is the most sensitive for SARS-CoV-2 detection.
In general, the simplicity of Chelex 100 Resin-based methods eliminates the need for additional plastic consumables associated with commercial kits, thus further reducing cost and waste.
Chelex 100 Resin and InstaGene Matrix are non-toxic, and Chelex 100 Resin offers stability with a long shelf life (3 years at room temperature). Thus, Chelex 100 is easy to transport and store, providing significant advantages when facing supply chain issues or the challenge of in-field testing. And, as Guan et al. indicates, SARS-CoV-2 RNA remains stable at room temperature after preparation using Chelex 100 Resin, further extending its applicability in the field, especially in remote and underdeveloped regions.
Chelex 100 Resin and InstaGene Matrix are attractive alternatives to commercial RNA extraction kits for several reasons. Studies on SARS-CoV-2 detection demonstrate that Chelex 100 Resin–based methods are well suited both for molecular detection where high sensitivity is critical and for surveillance applications where testing frequency and throughput are key. Chelex 100 Resin–based methods can accelerate molecular testing workflows, increase laboratory safety, minimize use of plastics, and reduce costs. These characteristics make it appealing not only for development of SARS-CoV-2 testing workflows, but also for routine monitoring of other infectious diseases, both now and in the future.Visit our website to learn more about the use of Chelex 100 Resin and Instagene Matrix for RNA extraction.
Chelex 100 Resin and InstaGene Matrix products are for research use only.
Flynn MJ et al. 2020. A simple direct RT-LAMP SARS-CoV-2 saliva diagnostic. medRxiv. Preprint. https://doi.org/10.1101/2020.11.19.20234948, accessed November 19, 2021.
Guan B et al., 2021. Sensitive extraction-free SARS-CoV-2 RNA virus detection using a chelating resin. iScience 24: 102960.
Howson ELA et al. 2021. Preliminary optimisation of a simplified sample preparation method to permit direct detection of SARS-CoV-2 within saliva samples using reverse-transcription loop-mediated isothermal amplification (RT-LAMP). J Virol Methods 289: 114048.
Lienhard A & Schäffer S. 2019. Extracting the invisible: obtaining high quality DNA is a challenging task in small arthropods. PeerJ 7: e6753.
Mardian Y et al. 2021. Review of current COVID-19 diagnostics and opportunities for further development. Front Med 8: 562.
Ulloa S et al. 2020. A simple method for SARS-CoV-2 detection by rRT-PCR without the use of a commercial RNA extraction kit. J Virol Methods 285: 113960.
Yaren O. et al. 2021. Ultra-rapid detection of SARS-CoV-2 in public workspace environments. PLOS One 16: e0240524.