key: cord-0850882-fifr7k99
authors: Page, Robert; Scourfield, Edward; Ficarelli, Mattia; McKellar, Stuart; Lee, Kwok Leung; Maguire, Thomas J.A.; Bouton, Clement; Lista, Maria Jose; Neil, Stuart J.D.; Malim, Michael H.; Zuckerman, Mark; Mischo, Hannah E.; Martinez-Nunez, Rocio T.
title: Homebrew: an economical and sensitive glassmilk-based nucleic-acid extraction method for SARS-CoV-2 diagnostics
date: 2022-03-03
journal: Cell Rep Methods
DOI: 10.1016/j.crmeth.2022.100186
sha: d136b1e39065d5a63ea963cb6146a1142af85a1a
doc_id: 850882
cord_uid: fifr7k99

Management of COVID-19 and other epidemics requires large-scale diagnostic testing. The gold standard for SARS-CoV-2 infection remains reverse transcription quantitative polymerase chain reaction (RT-qPCR)–analysis, which detects viral RNA more sensitively than any other method. However, the resource-use and supply chain requirements of RT-PCR have continued to challenge diagnostic laboratories worldwide. Here, we establish and characterise a low-cost method to detect SARS-CoV-2 in clinical combined nose and throat swabs allowing for automation in high throughput settings. This method inactivates virus-material with sodium dodecyl sulphate (SDS) and uses silicon dioxide as the RNA-binding matrix in combination with sodium chloride (NaCl) and isopropanol. With similar sensitivity for SARS-CoV-2 viral targets, but a fraction of time- and reagent expenditure compared to commercial kits, our method also enables sample pooling without loss of sensitivity. We suggest that this method will facilitate more economical widespread testing, particularly in resource-limited settings.

H o m e b r e w : S D S , N a C l , i s o p r o p a n o l , g l a s s m i l k , e t h a n o l , w a t e r

As the world approaches the 21 st month since COVID-19 was declared a pandemic by the World Health Organisation, global infections are still uncontrolled. Even in countries where combined vaccination and non-pharmacological strategies to contain the virus have decreased hospitalisation and death rates, new SARS-CoV-2 variants are causing local outbreaks. Therefore, widespread testing and contact tracing will remain as key tools for control of SARS-CoV-2 for the foreseeable future.

The gold standard for SARS-CoV-2 diagnostics consists of viral RNA extraction from combined nose and throat swab samples followed by RT-qPCR (PCR hereafter) . Standard RNA extraction for SARS-CoV-2 diagnostics is usually carried out with commercial kits, typically based on solid-phase reversible immobilisation (SPRI) of nucleic acids. While these kits are optimised to purify high-quality RNA for extremely sensitive downstream applications, in resource limiting situations their high cost per sample and requirement for dedicated supply chains and expertise can be a barrier to more widespread testing. Purification-free methods such as SalivaDirect, hid-RT-PCR, SwabExpress or others (Smyrlaki et al., 2020; Srivatsan et al., 2021; Vogels et al., 2021) , reduce time and costs, but compromise in sensitivity and/or require optimisation (e.g., depending on sampling buffer). Rapid methods have shown an increase in sensitivity when a rapid purification step is included (Joung et al., 2020) . Antigentesting with lateral flow devices provides a fast alternative solution, but such tests only detect peak infectivity with high confidence and are recommended for use in symptomatic individuals (Dinnes et al., 2021; Pickering et al., 2021) . These methods are therefore less suited for testing samples containing low viral loads such as those present at the initial stages of infection, or for population-wide screening of asymptomatic individuals when and where other more sensitive methods are available.

Prevention of outbreaks therefore requires highly sensitive methods that allow early detection. Thus, to control transmission, but also for variant detection, RNA isolation and PCR testing will continue to be irreplaceable.

While assessing different commercial kits for SARS-CoV-2 (Lista et al., 2021) we encountered bottlenecks in sourcing reagents, also reported elsewhere (Esbin et al., 2020) . We sought to establish an economical, scalable, and simplified method of RNA extraction circumventing worldwide shortages in reagents. Here we describe a method using only NaCl, SDS, isopropanol and ethanol, present in most laboratories worldwide, for the rapid extraction of SARS-CoV-2-RNA from combined nose and throat swab samples using silica powder ('glassmilk' or GM) as SPRI matrix. GM-based nucleic acid isolation was originally described in the 1970s (Boyle and Lew, 1995; Vogelstein and Gillespie, 1979) , employed later on for sequencing (Dederich et al., 2002) and also SARS-CoV-2 RNA isolation combined with RT-LAMP (Rabe and Cepko, 2020) . Developing an adjusted purification method, we established that GM can be replaced with carboxy magnetic beads that allow for automation. We provide a full characterisation of the limits of this method and demonstrate that the GM purification method allows for sample pooling without loss of sensitivity, as well as reduced plastic consumption. GM-nucleic acid purification could therefore contribute significantly to resource saving and supply-chain management in a world that will require long-term testing facilities for testing for SARS-CoV2 and other emerging viruses.

We set out to establish an alternative RNA extraction method for SARS-CoV-2 testing that would free our setting from supply-chain shortages. Figure 1A summarises the five optimised steps of our GM method that takes about 15 minutes per sample to perform (about 20 minutes per 24 samples). In the following we describe how we established and stress-tested this method using heat-inactivated combined nose and throat swabs in comparison to the gold standard rated (Centers for Disease Control and Prevention, CDC), SPRI-based QIAamp Viral RNA Mini kit (QIAamp hereafter). As combined nose and throat swabs can greatly differ (e.g. viscosity, material, pH, presence of inhibitors…) employing clinical samples when developing new methods, rather than virus-spiked viral transport media (VTM), is paramount to establish reliability, sensitivity and specificity of new methods.

Nucleic acids are known to reversibly bind silica in the presence of chaotropic salts and nonbasic pH conditions (Tan and Yiap, 2009) . While most commercial RNA extraction kits employ silica-coated magnetic beads or silica-based columns, we opted to use a suspension of silica particles as a solid matrix for RNA extraction. This suspension, also known as 'glassmilk' (GM), is economic and prepared by simply resuspending HCl-washed silica particles in water. GM particles are large enough to be efficiently pelleted by pulse-centrifugation, enabling separation of the solid and liquid phases during extraction.

Most commercial RNA extraction kits employ Guanidinium Thiocyanate (GITC) as chaotropic and protein denaturing salt to induce binding of nucleic acids to silica. However, the global shortage of GITC combined with its toxicity prompted us to test the capacity of different chaotropic salts to support silica binding. Both NaI and NaCl could substitute for GITC to support similar binding of viral RNA to GM (Figure 2A and S1A). It should be noted that in 3 of 6 combined nose and throat swabs SARS-CoV-2 RNA was also detected in the absence of chaotropic salts (water sample), presumably as heat inactivation or chaotropic salts contained within the VTM (containing Hanks Balanced Salt Solution) may cause some viral particles to disassemble (Smyrlaki et al., 2020) ; RNAseP was also detectable without major changes, potentially as lysed cells will release highly abundant RNAseP RNA. As a ubiquitously available and inexpensive salt, we chose NaCl as the chaotropic salt for future experiments.

To improve viral particle solubilisation from combined nose and throat swabs concomitant with inactivation (Patterson et al., 2020) we compared different detergents applied to the swabs. 100 µl of either 4% SDS, 4% Igepal, 4% Tween20, 4% Triton-X100 or water were added to 100 µl of swab sample, and the GM protocol was followed afterwards. Neither detergent changed the detection sensitivity with a clear, reproducible trend ( Figures 2B and S1B ). Since SDS showed the lowest mean difference to QIAamp (Ct = -2.579 for N1 and Ct = -2.762 for N2) and has been shown to inactivate SARS-CoV-2 at concentrations as low as 0.5% (Patterson et al., 2020) , we included it in our protocol.

Chaotropic salt-mediated nucleic acid precipitation and SPRI binding is improved in the presence of low dielectric constant solvents such as isopropanol, reviewed in (Tan and Yiap, 2009 ). Consistently, our protocol greatly increased test sensitivity in the presence of 50 % 2propanol ( Figures 2C and S1C ). We also assessed the need for ethanol washes, which showed that at least one 70% ethanol wash should be applied to the GM to remove protein contamination, increasing sample purity and thus improving detection (Figures 2D and S1D).

One advantage of GM is its fast separation under low g-forces enabling the use of cheap benchtop minicentrifuges or ultralow cost devices (Bhamla et al., 2017; Li, 2020) . We found that 7 mg of glass beads, corresponding to 10 µl of a 700 mg/ml GM solution, provided reproducible results and clean pelleting of the GM after 15 seconds of a pulse spin at 4500 x g ( Figures 2E and S1E ).

Most silica-matrix SPRI protocols depend on a narrow range of pH of sample material for efficient binding (Tan and Yiap, 2009 ). Our GM protocol performed robustly over a pH range of 6.5 to 10 ( Figures 2F and S1F ). In combination, GM was able to reliably isolate SARS-CoV-2 (PHE isolate 1) diluted into VTM up to a one plaque-forming unit per ml (pfu/mL) ( Figure  2G ), a limit of detection comparable to that of QIAamp. Human RNase P has been suggested as RT-PCR positive control by the CDC. To assess the reliability of RNaseP RNA isolation, we subjected serial dilutions of BEAS-2B bronchial epithelial cells resuspended in VTM and spiked with constant amounts of SARS-CoV-2 to the GM protocol. Figure 2H shows linear detection of RNaseP. The detection of SARS-CoV-2 in these samples was affected, although still detectable, at dilutions of 12,500 cells or less.

Finally, we stress-tested the GM protocol by inclusion of blood in combined nose and throat samples (20 L in 80 L of swab). Blood only marginally affected N2 detection but not N1 and all samples remained positive ( Figure S2A ).

Having established the individual components and limits of the GM protocol, we considered it crucial to reduce handling time and plastic usage to enable faster and cheaper high throughput testing. High throughput facilities employ commercial or open source automated stations that rely on magnetic bead isolation (Lazaro-Perona et al., 2021) . To this end, we prepared an extraction buffer that contained GM, 1.25M NaCl and isopropanol (GM_MB, GM master buffer), and compared this to the method employed thus far where we added the components separately (GM). These two methods did not show statistical differences in the Ct values of the samples ( Figure 3A , compare GM vs GM_MB). In addition, we prepared extraction buffers in which we exchanged GM for carboxy-coated magnetic beads (CB), which enable automation. Compared with GM, CB showed no statistical difference ( Figure 3A ).

Increased throughput for testing can also be achieved via sample pooling (Hogan et al., 2020) . This typically consists of mixing an equal volume of different samples, taking a representative volume, and analysing it in a single extraction and PCR (Lim et al., 2020) . Employing columnbased methods restricts pooling to small volumes with the consequent loss in sensitivity particularly for samples with high Ct values. Others have suggested concentrating swabs prior to extraction and PCR (Sawicki et al., 2021) . Although more sensitive, this increases processing time and costs for additional spin columns. As both GM_MB as well as CB_MB are based on solid matrices, either protocol allows scaling of the extraction for both manual and automated handling. To test this, we performed RNA extraction and PCR and compared Ct values obtained from five pools of samples that included one positive sample at different Ct values and 19 negative samples each ( Figure 3C ). 100 µl of a positive sample was either extracted individually (Sample) or pooled with 19 negative samples (100 µl each). RNA from pooled samples was extracted in two different ways.100 µl representative sample was taken from the pooled samples (Rep Pool) and extracted via the standard extraction protocol. Alternatively, we scaled SDS, NaCl and isopropanol proportionally and extracted RNA from the entire sample volume (Pool). It should be noted that the pooled extraction method only required GM for two reactions, i.e. 14mg, making it a cost savings of 0.0396 per 20 samples (plus plastics) and significantly reducing the handling time. Figure 3B shows that for GM the 'Pool' Ct did not statistically significantly change compared to 'Sample' Ct, while the 'Rep Pool' showed a significantly increased Ct for all pools. In the case of CB, both 'Pool' and 'Rep Pool' showed an increase in Ct values of -2.996 and -3.789, respectively. Importantly, none of our pools was rendered negative, which shows the feasibility of using both GM and CB in pooled samples.

As a final comparison, we employed 100 clinical specimens and compared GM and automated CB to QIAamp ( Figure 3C ). Our data showed similar sensitivity for N1 primer-probes between QIAamp and GM (98% vs 96%) while CB showed lower sensitivity (88%). To further show the reliability of this method, samples with > 5 Ct difference to QIAamp were highlighted (filled lines, 9 % GM, 32 % GB). Considering swabs with a viral load corresponding to 10 pfu/mL (Ct = 30 for QIAamp based on Figure 2G ), CB showed a sensitivity of 97% and GM 100% for N1 detection. N2 primer-probes showed a slightly decreased sensitivity to that of N1: 86% (CB), 97% (QIAamp) and 95% (GM), which was increased to 98% (CB) and 100% (GM) in samples with a QIAamp Ct above 30. The approximate price per reaction of both GM and CB is summarised in Figure 3D , considering list prices of representative suppliers in the UK. Our estimate is that costs per extraction are reduced around 40-fold for individual samples and ~400-fold for pooled samples.

In summary, we present a scalable, inexpensive and simple method that uses reagents widely available in laboratories worldwide to detect viral genetic material from combined nose and throat swabs with high sensitivity.

Glass -milk based isolation was initially described as cheap alternative for plasmid preparations (Boyle and Lew, 1995; Vogelstein and Gillespie, 1979) . However, with our current protocol, GM binds RNA preferentially, since recovery of a lentiviral plasmid was very poor (~1%, Figure S2B ), while that of small RNA (tRNA, ~90nt in length, Figure S2C ) and cellular RNA ( Figure S2D ) was much greater.

Our ability, to amplify RNase P in all nose and throat samples in our study (Figures 2, 3 and S1 G-L), attests to the GM protocol's capability of isolating cellular RNA. However, the quality of that RNA is low ( Figure S2 C), and ribosomal RNA appears degraded. We therefore note that whilst it might be possible to use GM for cellular RNA isolation, the current protocol will require further adjustment. From preliminary experiments we can say that cell lysis seems a key step in the process, since recovery of material was improved when cells were resuspended in PBS and lysed in a final 2% SDS concentration vs cells directly lysed in 4% SDS ( Figure  S2Ci ). This was potentially also cell type specific, since two different cell lines (A549 and BEAS2B) rendered very different qualities ( Figure S2Cii ). Despite the suboptimal RNA quality, we were able to detect GAPDH and IL8 mRNAs by qPCR with a limit of 250,000 cells per reaction, below which Ct values remained too high ( Figure S2Ciii ). This is in line with our data in Figure 2H , where we observed a loss in sensitivity for N1 and N2 detection when there was less RNA present in the samples. As SARS-CoV-2 detection decreases with lower amounts of cell-input, we surmise that cellular RNA may act as carrier RNA, facilitating SARS-CoV-2 RNA binding to GM. Moreover, the DNA and RNA extracted employing homebrew GM did not contain DNAse or RNAse activity, since incubation of GM-isolated DNA or RNA with exogenous DNA and RNA did not cause observable degradation ( Figure S2D ). Thus, homebrew GM has the potential of being used to perform cellular RNA extractions, provided appropriate cellular lysis conditions.

Multiple novel approaches have been developed to evaluate SARS-CoV-2 infection (Eftekhari et al., 2021; Kevadiya et al., 2021) . Different test types (e.g. antigen tests; serological tests) have their place according to needs and resources; however, PCR remains the most sensitive method and the gold standard to which all tests compare to. We believe that our method is of value currently, as cases continue to soar in many parts of the world. The current pandemic has taught us that future zoonotic transmission is highly likely to cause outbreaks. Cheaper and simpler viral detection methods will therefore be continuously required. We thus believe that the method presented here has longstanding potential in viral disease control.

We acknowledge that our study comes with limitations. Our experiments were performed on swabs that had been tested for the presence of SARS-CoV-2 and were subsequently frozen; the influence of freeze-thawing material cannot be measured in our study. In order to make our protocol useful for others we introduced a first step of heat inactivation of swab material, as many laboratories face the problem of dealing with non-inactivated material. It is possible that the heat inactivation allows better performance of the master buffer made with waterand hence addition of detergents does not seem to strongly influence Ct values (Figures 2B and S1B 

No authors declare any competing interests.

The author list of this paper includes contributors from the location where the research was conducted who participated in the data collection, design, analysis, and/or interpretation of the work.

J o u r n a l P r e -p r o o f 

A) Chaotropic salt titration: Viral RNA extracted from six combined nose and throat swabs using different chaotropic salts, 2-propanol and 7 mg GM, shows in a one-way ANOVA test significant increase in Ct values compared to the bench-mark QIAamp viral RNA mini kit (P-adj < 0.0001 for water, NaCl, NaI, Guanidiumthiocyanate (GITC). NaCl and NaI had 100 % sensitivity, whereas. GITC and water lost one and three samples respectively. B) Detergent titration: Addition of different detergents prior to RNA extraction from ten combined nose and throat swabs, shows a significant difference to QIAamp (adjusted P-adj one-way ANOVA of < 0.0001. All detergents but Tween-20 have been shown to inactivate SARS-CoV-2 at 1 % (Patterson et.al., 2020 QIAamp vs 3.75 P -adj = 0.0114 and QIAamp vs 1.875 P -adj < 0.0001 F) Effect of pH of the RNA-extraction buffer on detection of viral RNA from 6 combined nose and throat swabs compared to QIAamp: pH 4.5 (P-adj < 0.0001), pH 6.5 (ns = 0.3709), pH 6.75 (ns = 0.2187), pH 8.1 (ns = 0.2433) and pH 9 (ns = 0.5434). G) Level of detection of QIAamp, compared to the homebrew method using GM and tenfold serial dilutions of SARS CoV-2 in viral transport medium. Ct= Cycle threshold, pfu = plaque forming unit. The dilution series was best fit with a non-linear regression. H) Level of detection of RNAseP, N1 and N2 employing homebrew GM depending on cell numbers present in the sample. Ct= Cycle threshold. BEAS-2B cells were prepared at different numbers and spiked with equal amounts of SARS-CoV-2, and assessed using GM. The dilution series was best fit with a linear regression. J o u r n a l P r e -p r o o f

Resource Availability:

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Rocio T. Martinez-Nunez rocio.martinez_nunez@kcl.ac.uk.

This study did not generate new unique reagents.

• RT-qPCR data have been deposited at Mendeley at https://data.mendeley.com/datasets/b2mscbnhmg/2, and are publicly available as of the date of publication. The DOI is listed in the key resources table. • This paper does not report original code.

• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Delivery for King's College Hospital. All samples were combined nose and throat swabs having already been tested at King's College Hospital or KCL TEST as part of a potential service development. All samples were anonymised and assessed after being diagnosed as SARS-CoV-2 positive by the hospital or negative by KCL TEST. Samples were chosen based on Ct values in order to represent a broad range of values. Positive swabs were inactivated in a Category 3 facility employing 90°C 10 mins in a dry bead bath (Lista et al., 2021) .

Glass milk preparation Important note: work under fume hood. Suspend silicon dioxide 325 mesh (Sigma 342890) in 40mL 10% HCl from a 37% HCl stock diluted in water in a 50mL tube, agitate suspension for 4 hrs on a tube roller and centrifuge at 2000 g. Remove supernatant and resuspend the silica pellet with 40mL water. Centrifuge and repeat the water wash five times (a total of six washes). Make sure the silica is fully resuspended after each wash so residual HCl is not 'trapped' in the pellet mass. After the final wash, weigh the pellet and resuspend in water at 700 mg/mL. The pH of the silica should be between 7 and 8.

VTM preparation Viral transport medium was prepared as per CDC instructions (https://www.cdc.gov/coronavirus/2019-ncov/downloads/Viral-Transport-Medium.pdf), i.e. 2% FBS 100µg /mL Gentamicin 0.5 µg /mL Amphotericin B in Hanks Balanced Salt Solution (HBSS).

Combined nose and throat swab samples were heat-inactivated at 90C for 10 min in a Cat-3 facility. Swabs that did not contain enough volume for all comparisons were topped up with J o u r n a l P r e -p r o o f negative combined nose and throat swabs in viral transport medium. In a 1.5 ml tube, 100µl of sample were mixed with 100µl of 4% SDS. 610 µl of glassmilk extraction buffer (GM-MB) (200 µl 1.25 M NaCl, 400 µl 2-propanol and 10µl of GM) were added to each sample, the mixture was inverted, vortexed and incubated at room temperature for 5 minutes. The sample was then centrifuged for 15 seconds in a bench-top microfuge at 4600 x g and the supernatant poured off. The silica pellet was resuspended in 500 l of 70 % ethanol before being centrifuged for 15 seconds at 4600 x g (wash 1). The supernatant was poured off and the silica pellet was resuspended again in 500 l of 70 % ethanol and centrifuged for 15 seconds at 4600 x g (wash 2). After another spin at 4600 x g (15 sec), all remaining ethanol in the supernatant was carefully removed with a pipette and the silica pellet was air-dried at 65C for 5 minutes. Finally, RNA was eluted by resuspending the silica pellet in 50µl of nuclease-free water. The tube was centrifuged for 15 seconds and the supernatant containing RNA was transferred to a fresh 1.5ml tube.

Adaptation to magnetic beads All steps as above, except that the GM-EB contained 10 µl 50 mg/ml carboxylate modified magnetic SpeedBeads (Merck GE45152105050250). To collect beads manually, we employed a magnetic rack (Invitrogen, DynaMag™-2 Magnet, 12321D). Beads were dried for 5 minutes at room temperature. Figure 2A : testing different salts. The same protocol as described above was used, except that instead of GM-EB, we added its components separately to allow assessment of individual chaotropic salts: 100l of sample was mixed with 100l water to which 200 µl of either 1.25M NaCl, 1.25M NaI, 1.25M GITC or water were added. 10l of GM [700mg/ml] and 400l of isopropanol was added before incubating the mixture at room temperature for 5 minutes. Figure 2B : testing different detergents. We mixed 100 µl of combined nose and throat swabs with 100 µl of either 4 % SDS, 4 % Igepal, 4 % Tween, 4 % Triton-X100 or water, before 200 µl 1.25 M NaCl, isopropanol and 10l of GM [700 mg/ml] were added and the standard protocol followed thereafter. Figure 2F : assessment of pH ranges. 100 µl swab material was inactivated for 10 min at 90 C before 100 µl of 4 % SDS were added. NaCl solutions were buffered with hydrochloric acid to different pH so that mixing with the swab would yield the pH indicated in the figure: NaCl buffered to pH1.5 results in pH 4.5 after mixing; NaCl buffered to pH3, 6.75, 10.75 and 11 resulted in swabs with pH 6.5, 6.75 8.1 and 9.8, respectively, after mixing. Figure 3B Pooling: 100 µl heat-inactivated swab material (10 min at 90 C) from 20 different samples were mixed in a 50mL tube. To this mixture, 2mL of 4 % SDS, 4mL of 1.25 M NaCl, 8mL of isopropanol and 20µl of GM were added. Samples were mixed and spun, washed twice with 1mL 70% ethanol in a 1.5mL tube and the GM pellet was air dried at 65  C for 5 min prior to resuspending in 50µl of nuclease free water. inactivated sample was used as input material and 50 l of nuclease-free water was used to elute RNA from the column.

Plasmid DNA purified in Figure S2B was that of pLVTHM_shRNA control (Martinez-Nunez et al., 2017) .

RT-qPCR RT-qPCR reactions were carried out using TaqMan Fast Virus 1-Step Master Mix (ThermoFisher Scientific, 4444434). Primer-probe sets for used SARS-CoV-2 detection were the CDC-recommended sets N1, N2 and RNaseP Emergency Use Authorisation kit (Integrated DNA Technologies, 2019-nCov CDC EUA Kit, 10006770). RT-qPCR reactions containing 5l master mix, 1.5l pre-mixed primer-probe, 8.5l water and 5l purified RNA were run on a QuantStudio 5 (Applied Biosystems/ThermoFisher Scientific) using the "Fast" cycling mode. Figure S2 experiments were run on a QuantStudio 7 Flex (Applied Biosystems/ThermoFisher Scientific). The cycling conditions used were 50°C 5 mins, 95°C 20 sec, and 45 cycles of denaturation (95°C, 3 sec) and annealing/extension (60°C, 30 sec). RT-qPCR reactions were performed in duplicate for all samples, except for those used in Figure 3B to resemble a testing setting and done in singlets. Undetermined samples were set at a Ct of 40 for statistical and representation purposes, except in the GM titration figure ( Figure 2F ) where we considered values above 40 to stress-test the system. Figure S2Ciii was done in two steps. 150ng of RNA were reverse transcribed with RevertAid H Minus Reverse Transcriptase (ThermoFisher Scientific) using 100pmol random hexamers in a final volume of 10 L.(25°C, 10 min; 42°C, 60 min; 70°C, 10 min). 1:5 diluted complementary DNA (cDNA) was amplified by qPCR using GAPDH (Primer Design) and IL6 (ThermoFisher Scientific) primers and the Luna® Universal Probe qPCR Master Mix (New England Biolabs). 1 L of diluted cDNA was mixed with 5 L master mix buffer, 0.25 L primer-probe mix and 3.75 L of nuclease free water in a final reaction of 10 L. Cycling parameters (in fast mode) were 95°C 60 sec, and 45 cycles of denaturation (95°C, 15 sec) and annealing/extension (60°C, 30 sec).

Statistics were performed on the Ct values obtained by amplification using the N1 primerprobe sets in Figures 2 and 3 and N2 and RNAseP in Figure S1 . All datasets were initially assessed for normality distribution using a Kolmogorov-Smirnov test. Parametric data were compared using a one-way ANOVA with a multiple correction Dunnett's test. Non-parametric data were analysed using a Friedman test with Dunn's multiple comparison test. Paired t-tests were employed in Supplemental Figure S2A . Reported P-values are p-adjusted values from the multiple comparison tests. P-value was deemed significant when p-adjusted was < 0.05. *: Padjusted < 0.05; ** P-adjusted < 0.01; ***: P-adjusted <0.001; ****: P-adjusted < 0.0001 A) and G). Chaotropic salt titration: Viral RNA extracted from six combined nose and throat swabs using different chaotropic salts, 2-propanol and 7 mg GM, shows in a oneway ANOVA test significant increase in Ct values compared to the bench-mark QIAamp viral RNA mini kit. B) and H). Detergent titration: Addition of different detergents prior to RNA extraction from ten combined nose and throat swabs, shows a significant difference to QIAamp (adjusted P-adj one-way ANOVA of < 0.0001. All detergents but Tween-20 have been shown to inactivate SARS-CoV-2 at 1 % (Patterson et.al., 2020) . C) and I). Isopropanol effect on GM binding. Friendman's test with Dunn's multiple comparison showed non-significant differences by adjusted P-adj for N2 but a loss in sensitivity in detecting RNAseP as per ANOVA analysis. D) and J). Matrix wash with Ethanol increases sensitivity. One-way ANOVA test shows no significant differences to the QIAamp viral RNA mini kit (D) and Friendman's test with Dunn's multiple comparison (J) showed loss of detection of RNAseP for one sample. E) and K). GM quantities. ANOVA test with Dunnett's multiple comparison test vs QIAamp showed no difference in N2 detection using 7mg GM vs QIAamp and P-adj 0.0021 for RNAseP detection (mean difference -1.729). F) and L). Effect of pH of the RNA-extraction buffer on detection of viral RNA from 6 combined nose and throat swabs compared to QIAamp. OnlypH 4.5 showed a significant difference vs QIAamp for N2 detection (F, P-adj < 0.0001) while detection of RNAseP (L) appeared not affected. , pH 6.5 (ns = 0.3709), pH 6.75 (ns = 0.2187), pH 8.1 (ns = 0.2433) and pH 9 (ns = 0.5434). A) Effect of blood in sensitivity of detection of SARS-CoV-2. 6 negative combined nose and throat swabs were spiked with SARS-CoV-2 and heat inactivated, and blood was added (or not). Sensitivity of N1 detection appeared not affected, with N2 increasing in Ct marginally (P = 0.0136). Paired t-tests were employed. More RNAseP was detected when swabs were spiked with blood, as expected. B) Recovery of plasmid DNA (left), small RNA (middle, tRNA, ~90nt) and cellular RNA (right) by homebrew GM. C) Recovery of cellular RNA from cultured cells depending on lysis conditions (i) as measured by Qubit, RNA integrity (ii) and Ct values of GAPDH and IL8 in 150ng of RNA isolated from different cell numbers (iii). D) DNAse (left) and RNAse (right) assessment of GM-isolated extracts. Plasmid DNA (left) or cellular RNA (right) were incubated with DNA or RNA extracted employing GM homebrew. . Chaotropic salt titration: Viral RNA extracted from six combined nose and throat swabs using different chaotropic salts, 2-propanol and 7 mg GM, shows in a one-way ANOVA test significant increase in Ct values compared to the bench-mark QIAamp viral RNA mini kit. B) and H). Detergent titration: Addition of different detergents prior to RNA extraction from ten combined nose and throat swabs, shows a significant difference to QIAamp (adjusted P-adj one-way ANOVA of < 0.0001. All detergents but Tween-20 have been shown to inactivate SARS-CoV-2 at 1 % (Patterson et.al., 2020) . C) and I). Isopropanol effect on GM binding. Friendman's test with Dunn's multiple comparison showed non-significant differences by adjusted P-adj for N2 but a loss in sensitivity in detecting RNAseP as per ANOVA analysis. D) and J). Matrix wash with Ethanol increases sensitivity. One-way ANOVA test shows no significant differences to the QIAamp viral RNA mini kit (D) and Friendman's test with Dunn's multiple comparison (J) showed loss of detection of RNAseP for one sample. E) and K). GM quantities. ANOVA test with Dunnett's multiple comparison test vs QIAamp showed no difference in N2 detection using 7mg GM vs QIAamp and P-adj 0.0021 for RNAseP detection (mean difference -1.729). F) and L). Effect of pH of the RNA-extraction buffer on detection of viral RNA from 6 combined nose and throat swabs compared to QIAamp. Only pH 4.5 showed a significant difference vs QIAamp for N2 detection (F) while detection of RNAseP (L) appeared not affected. negative combined nose and throat swabs were spiked with SARS-CoV-2 and heat inactivated, and blood was added (or not). Sensitivity of N1 detection appeared not affected, with N2 increasing in Ct marginally (P = 0.0136). Paired t-tests were employed. More RNAseP was detected when swabs were spiked with blood, as expected. B) Recovery of plasmid DNA (left), small RNA (middle, tRNA, ~90nt) and cellular RNA (right) by homebrew GM. C) Recovery of cellular RNA from cultured cells depending on lysis conditions (i) as measured by Qubit, RNA integrity (ii) and Ct values of GAPDH and IL8 in 150ng of RNA isolated from different cell numbers (iii). D) DNAse (left) and RNAse (right) assessment of GM-isolated extracts. Plasmid DNA (left) or cellular RNA (right) were incubated with DNA or RNA extracted employing GM homebrew. 

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Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation

Methods of Inactivation of SARS-CoV-2 for Downstream Biological Assays

Comparative performance of SARS-CoV-2 lateral J o u r n a l P r e -p r o o f flow antigen tests and association with detection of infectious virus in clinical specimens: a single-centre laboratory evaluation study

SARS-CoV-2 detection using isothermal amplification and a rapid, inexpensive protocol for sample inactivation and purification

Sample pooling as a strategy for community monitoring for SARS-CoV-2

Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR

SwabExpress: An end-to-end protocol for extraction-free covid-19 testing

DNA, RNA, and protein extraction: the past and the present

SalivaDirect: A simplified and flexible platform to enhance SARS-CoV-2 testing capacity

Preparative and analytical purification of DNA from agarose