SARS-CoV-2 detection using reverse transcription strand invasion based amplification and a portable compact size instrument

Rapid nucleic-acid based tests that can be performed by non-professionals outside laboratory settings could help the containment of the pandemic SARS-CoV-2 virus and may potentially prevent further widespread lockdowns. Here, we present a novel compact portable detection instrument (the Egoo Health System) for extraction-free detection of SARS-CoV-2 using isothermal reverse transcription strand invasion based amplification (RT-SIBA). The SARS-CoV-2 RT-SIBA assay can be performed directly on crude oropharyngeal swabs without nucleic acid extraction with a reaction time of 30 min. The Egoo Health system uses a capsule system, which is automatically sealed tight in the Egoo instrument after applying the sample, resulting in a closed system optimal for molecular isothermal amplification. The performance of the Egoo Health System is comparable to the PCR instrument with an analytical sensitivity of 25 viral RNA copies per SARS-CoV-2 RT-SIBA reaction and a clinical sensitivity and specificity between 87.0–98.4% and 96.6–98.2% respectively.

Here we present a new compact size portable point of care (POC) system called the Egoo Health System, which consists of an Egoo instrument (470 g), an Egoo clinical application (app), and an Egoo assay capsule. The Egoo Health System was initially designed for detection of biomarkers in blood, saliva and urine, but here we show that the Egoo Health System can also be used to detect SARS-CoV-2 from oropharyngeal swabs. The compact size Egoo instrument is designed for use with multiple assay types, each utilizing a single-use, test specific assay capsule, thus the Egoo Health System can be used for several different assays, including both biochemical and NAAT tests. After an assay run, raw data is sent via WiFi to the Egoo server for raw data analysis, and the calculated assay result is sent to a laptop/smartphone, which is the primary display for the Egoo Health System. The Egoo Health System was initially developed for use in private homes but can also be used in primary care clinics, nursing homes, and workplaces, without the need of specialized laboratory staff.
For detection of SARS-CoV-2 with the Egoo Health system we developed an SARS-CoV-2 Egoo capsule based on the previously described reverse transcription strand invasion based amplification (RT-SIBA) isothermal technology and SYBR green detection [28][29][30][31] . The SARS-CoV-2 RT-SIBA assay can be performed directly on crude oropharyngeal swabs without NA extraction with a reaction time of 30 min.

Methods
Ethical statement. The study is not a medical health science research project but a method comparison study using surplus material from routine oropharyngeal samples collected at Nordsjaellands hospital and Amager and Hvidovre University hospital. The need for informed consent and ethical approval were reviewed by the Institutional Review Board at Amager and Hvidovre University Hospital and Nordsjaellands hospital and the regional research ethic committee for Region Hovedstaden (The National Committee on Health Research Ethics, Region Hovedstaden, Blegdamsvej 60, 1. Sal opgang 94A11, DK-2100 Copenhagen) and found not to need approval according to national ethic research regulation.
The Egoo Health System. The Egoo Health System comprises of an Egoo instrument (capsule reader), an Egoo power adaptor, an Egoo clinical application (app), and an Egoo assay capsule. The Egoo instrument is compact in size (W66 × H107 × D94 mm), and weight (470 g/1.04 lbs). It consists of an integrated optical microelectromechanical system (optical MOEM) 32 and has a dual optical unit for simultaneously measuring fluorescence and absorbance. The optical unit is integrated on a micro heating (max. 50 °C (122°F)) and vortex mixing unit (max. 3000 rpm) for heating and mixing the assay reagents during assay runs. The Egoo instrument has a mechanical piston mechanism which via a plunger unit can tightly seal the Egoo assay capsule after applying the sample, resulting in a closed system. In addition, the piston mechanism can inject various reagents from the integrated capsule injection chambers which sit on top of each assay specific Egoo capsule. Instrument specifications can be seen in supplementary Table S1. The SARS-CoV-2 Egoo capsule (W24 × H28 × D36 mm) (supplementary Table S2) consists of 140 µl frozen SARS-CoV-2 RT-SIBA Mastermix. The SARS-CoV-2 Egoo capsules are stored at − 20 °C until their intended use. Stability studies of the SARS-CoV-2 Egoo capsules are ongoing, but the current shelf-life of the SARS-CoV-2 Egoo capsule is 180 days at − 20 °C (supplementary Fig. S1).
Egoo data analysis. After each assay run, the raw data is sent via WiFi to the Egoo server for raw data analysis. For the SARS-CoV-2 RT-SIBA assay, the calculations are based on 3 fluorescence measurements recorded every minute for 30 min. The fluorescence measurements are graphed via an algorithm within the server. Based on an initial data set of > 100 positive/negative oropharyngeal patient samples used to define minimum and maximum values within the algorithm (data not shown), the assay result is determined. The primary display for the Egoo Health System is a laptop/smartphone. Based on the accumulated curve and slope, the assay result is reported as either "negative", "positive" or "inconclusive" to the end user. Alternatively, the raw data can be analysed via Excel or other graphing programs, should the end user wish to visualize the resulting amplification curve from the reaction. www.nature.com/scientificreports/ SARS-CoV-2 RT-SIBA. The SARS-CoV-2 RT-SIBA assay consist of a forward primer (5′-GAA CTT TAA GTC AGT TCT T-3′), reverse primer (5′-CAG TCT CAG TCC AACA-3′) and an invasion oligo containing a Cytosine 5′ overhang and 2′-O-methyl RNA moieties in the 3′end (5′-CCC CCC CCC CCC CCT TTA TTA TCA AAA  CAA TGT TTT TAT GTC TGAAG CAA AAT GTT -3′ 34 targeting the E-gene (Charité Berlin) 33  www.nature.com/scientificreports/ E-gene RT-PCR assay 33 from the Qlife COVID-19 Service Center were analyzed. Informed patient consent was obtained for Qlife patient samples. In addition, two independent method comparison studies were performed at two different hospitals. At Hvidovre and Amager hospitals, 700 retrospective oropharyngeal swabs dissolved in UTM previously diagnosed positive or negative for SARS-CoV-2 using the SARS-CoV-2 Roche Flow/MGI-BGI RT-PCR assay were re-tested followed by analysis using the SARS-CoV-2 Egoo capsule on the Egoo instrument. At Nordsjaellands hospital, 224 patient samples diagnosed positive or negative for SARS-CoV-2 using the SARS-CoV-2 Cobas Liat System (Roche) were re-tested within 24 h using the SARS-CoV-2 Egoo capsule on the Egoo instrument. All oropharyngeal patient swabs were collected in accordance with national guidelines and regulations and only surplus material from routine oropharyngeal samples were used in this study.

Results
The Egoo Health System. The Egoo Health System consists of a small Egoo instrument, a laptop or mobile phone, with the Egoo clinical app, and an Egoo capsule containing the assay of interest ( Fig. 1a-d). Because of the limited heating system in the Egoo instrument (max. 50 °C), we developed an isothermal SARS-CoV-2 RT-SIBA assay for the Egoo capsule that target the RdRp gene in SARS-CoV-2 genome (Fig. 1e). In silico analysis of 699,737 full-length SARS-CoV-2 sequences submitted to GISAID 35 Table S3). Only 0.37% of the sequences analysed contained SNPs in either the binding region of the primers or the invasion oligo. A representative figure of the observed SNPs can be seen in supplementary Fig. S4. For methodological simplification of the Egoo Health system, the SARS-CoV-2 RT-SIBA assay was designed to be used directly on crude samples without NA extraction using a SIBA lysis/reaction buffer (Fig. 1f), which contain mild detergents and magnesium for activation of the SARS-CoV-2 RT-SIBA assay.
The analytical specificity of the SARS-CoV-2 RT-SIBA assay. First, we wanted to investigate the analytical specificity of the SARS-CoV-2 RT-SIBA assay against other human coronaviruses (hCoV-NL63, hCoV-229E and hCoV-OC43) and the most common human respiratory viruses such as Influenza A H1N1, Influenza B and Respiratory Syncytial virus (RSV) ( Table 3). Purified viral NA or inactivated virus cultures from the different viruses were spiked into a SARS-CoV-2 negative oropharyngeal background. The samples were diluted 10-fold in SIBA lysis/reaction buffer before being analysed with the SARS-CoV-2 RT-SIBA assay using two different PCR instruments (MX3005P or CFX96) and the small Egoo instrument using SARS-CoV-2 Egoo capsules (Table 1). Additional specificity testing of the SARS-CoV-2 Egoo capsules against a wide range of common human viruses and bacteria can be seen in supplementary Table S4. No cross-reactivity to other respiratory viruses or bacteria was observed for either the purified NA or non-purified viral and bacterial cultures and the SARS-CoV-2 RT-SIBA assay was 100% specific for SARS-CoV-2.
The analytical sensitivity of the SARS-CoV-2 RT-SIBA assay. Next, we wanted to test the analytical sensitivity of the SARS-CoV-2 RT-SIBA assay. Synthetic SARS-CoV-2 RNA and inactivated SARS-CoV-2 virus culture was spiked into negative oropharyngeal swab at different concentrations and diluted 10-fold in SIBA lysis/reaction buffer. The different dilutions were tested using both a PCR instrument (Mx3005P) and five different Egoo instruments (Fig. 2a,b, Table 2). Figure 2 shows the amplification curves of the synthetic SARS-CoV-2 RNA from the PCR instrument (Fig. 2a) and five different non-calibrated Egoo instruments (Fig. 2b). For the PCR instrument, the dilutions were predominantly detected with 8-16 min, whereas the dilutions were detected within 12-22 min on the different Egoo instruments (Fig. 2, Table 2). For the endpoint fluorescence signal, differences between the Egoo instruments were observed which indicated that the Egoo instruments were not calibrated, however, this did not influence the calculated results from the backend server. The limit of detection (LOD) of the SARS-CoV-2 RT-SIBA assay was found to be between 20 and 25 RNA copies/reaction when using both the synthetic RNA and whole virus culture. No difference between the LOD was observed between the PCR instrument and the five Egoo instruments tested (Table 2). However, for the PCR instrument only 2.5 µl of sample was loaded into the PCR tube containing 17.5 µl SARS-CoV-2 RT-SIBA mastermix, whereas 20 µl of sample was loaded into the SARS-CoV-2 Egoo capsule (containing 140 µl of mastermix). This corresponds to a lower sample input concentration for the Egoo instrument compared to the PCR instrument. The SARS-CoV-2 Egoo Health system can detect as low as 1.3 viral RNA copies/µl (Table 2). www.nature.com/scientificreports/ Extraction free sample handling. The SARS-CoV-2 RT-SIBA assay was designed to be used directly on crude samples without NA extraction using a SIBA lysis/reaction buffer (Fig. 1f). To test if lysis can be performed directly in the sample collection tube, different concentrations of SARS-CoV-2 virus culture were spiked into a negative oropharyngeal swab background and subsequently added to sampling swabs (n = 16). SIBA lysis/reaction buffer (500 µl) was added directly to the tubes containing the spiked swabs and the tubes were incubated for 10 min at RT with shaking to release the viral RNA from the swab, before being analysed by the SARS-CoV-2 RT-SIBA assay using a PCR instrument. As negative controls, negative oropharyngeal swabs (n = 18) directly dissolved SIBA lysis/reaction buffer were also analysed (Fig. 3a). Swabs spiked with 4200 viral RNA copies and dissolved in SIBA lysis/reaction buffer to a concentration of 79 viral RNA copies could easily be detected with the SARS-CoV-2 RT-SIBA reaction (Fig. 3a). Analysis of SARS-CoV-2 negative oropharyngeal swabs showed positive detection in 39% (7/18) of the samples (Fig. 3a) and analysis of the melting curve showed a second melting peak around 56 °C, whereas the correct melting peak for a SARS-CoV-2 positive sample should be at 68 °C (Fig. 3b). The SARS-CoV-2 assay is based on a SYBR Green detection of the amplified product, and therefore any non-target amplification occurring due to the genomic background in the sample is also detected. This nontarget amplification is easily distinguishable from target-specific amplification in a PCR instrument by performing melt curve analysis. Since melt curve analysis cannot be performed in the Egoo instrument, we investigated if diluting the samples by 2-, 5-, 10-, 20-fold would reduce non-target amplification (Fig. 3c,d). Dilution of the  www.nature.com/scientificreports/ samples clearly showed that 10-and 20-fold dilutions of the samples eliminated the formation of the non-target melting peak (Fig. 3c,d). To further test this, 128 negative oropharyngeal patient swabs (dissolved in 1 ml PBS) www.nature.com/scientificreports/ were 10-fold diluted in SIBA lysis/reaction buffer and analysed with the SARS-CoV-2 RT-SIBA assay. Only 1/128 (0.7%) of the 10-fold diluted negative oropharyngeal swabs showed non-target amplification, with a very small amplification curve (Fig. 3e). These results show that samples must be diluted 10-fold in SIBA lysis/reaction buffer before being analysed on the Egoo instrument. To methodologically simplify the dilution procedure for non-professionals, 10 ml of SIBA lysis/reaction buffer must be added to a sample collection tube if the sample is to be used directly with the SARS-CoV-2 Egoo capsule and the Egoo instrument (Fig. 3f).
Compatibility to sample media. Since samples must be diluted 10-fold to reduce non-target amplification in the oropharyngeal swab we wanted to investigate whether other sampling media such as UTM and VTM can be used with the Egoo Health System. We also tested different media used for antigen quick tests. We simulated SARS-CoV-2 positive samples by spiking SARS-CoV-2 virus culture (Hong Kong/VM2000i06i/2020 or (USA-WA1/2020)) into PBS, UTM (Copan), VTM (Nest Biotechnology), VTM (Mole Bioscience), Gensure antigen buffer, Acro antigen buffer, Biosynex antigen buffer or SD Biosensor antigen buffer (1:10). The simulated samples were further diluted 10-fold in SIBA lysis/reaction buffer before the samples were analysed with the SARS-CoV-2 Egoo capsule on the Egoo instrument (Fig. 4). The SARS-CoV-2 RT-SIBA assay was not influenced by the presence of UTM (Copan), VTM (Nest Biotechnology), VTM (Mole Bioscience), and all dilutions of the virus culture (Fig. 4a,b) were detected except the SARS-CoV-2 negative samples (Fig. 4g). In addition, we observed no difference between SARS-CoV-2 positive samples dissolved in PBS and Acro antigen buffer, Biosynex antigen buffer or SD Biosensor antigen buffer (Fig. 4d-f). However, SARS-CoV-2 positive samples dissolved in Gensure antigen buffer were inhibited (Fig. 4c). These results show that the SARS-CoV-2 RT-SIBA assay is dependent on the sample collection media and only the above-mentioned medias have been validated for the SARS-CoV-2 Egoo capsule. Figure 4h shows an overview of the SARS-CoV-2 Egoo Health system when using other sampling media.
Sample stability in SIBA lysis/reaction buffer. The SARS-CoV-2 RT-SIBA reaction is dependent on the SIBA lysis/reaction buffer containing mild detergents and magnesium. The stability of SARS-CoV-2 in oropharyngeal samples when diluted 10-fold in SIBA lysis/reaction buffer was tested at different timepoints after  (Fig. 5). Samples stored at 5 °C in SIBA lysis/reaction buffer were stable up to 24 h (Fig. 5a,c). However, when stored at 30 °C the SIBA lysis/reaction buffer samples were stable up to 4 h for oropharyngeal PBS samples (Fig. 5b) and 8 h for oropharyngeal UTM samples (Fig. 5d). No difference in time to positive was observed for samples store at 5 °C compared to samples stored at 30 °C.
Comparison to reference RT-PCR assays and platforms. The direct lysis and 10-fold dilution of the sample could potentially result in a lower clinical sensitivity of the SARS-CoV-2 RT-SIBA assay. To test this, the performance of the SARS-CoV-2 Egoo capsule on the Egoo instrument was tested on patient samples diagnosed positive or negative for SARS-CoV-2 using different RT-PCR platforms. Two of the studies were performed independently at two different Danish hospitals. In total 1154 patient samples were tested with the SARS-CoV-2 Egoo Health system and compared to either; (1) direct lysis using SIBA lysis/reagent buffer (no NA purification) followed by RT-PCR for the E-gene 33    These results show that a 10-fold dilution of the sample in SIBA/reaction buffer will result in a lower clinical sensitivity compared to standard RT-PCR methods, however only samples with very high ct-values will not be detected with the SARS-CoV-2 Egoo Health System. The specificity of the SARS-CoV-2 RT-SIBA assay in the Egoo instrument ranged from 96.6 to 98.2% (Table 3) showing that the 10-fold dilution of the sample in SIBA lysis/reaction buffer significantly reduces the non-target amplification of the SYBR based assay. The results were obtained using the Clinical app, but if we evaluate the results by the slope of the amplification curves of the true positives and false positives, the big comparison study (n = 1154) showed a clear difference in the slope of the curves (Supplementary Fig. S5) which could indicate that adjustments to the current algorithm could increase the specificity of the SARS-CoV-2 RT-SIBA assay on the Egoo instrument to 98.2-100%.

Discussion
Here, we present for the first time a very compact instrument called the Egoo Health System, which has been developed for home-use monitoring of biochemical markers. The Egoo Health System is simple to use and can be used in private homes, primary care clinics, nursing homes, and workplaces without the need for specialized laboratory staff.
The Egoo Health System uses specialized Egoo capsules that are sealed in the Egoo instrument with a piston mechanism and a plunger. Once the plunger has sealed the capsule tight, the reaction begins which eliminates the risk of amplicon contamination. The amplification steps in NAAT tests are extreme, resulting in billions of copies of the target of interest. This amplification step requires a closed system to avoid amplicon contamination and the detection of false positives. Opening a tube after an amplification to use it on e.g. a lateral flow stick 19,36 is possible to do in a specialised laboratory but is not possible to do outside a laboratory in the current form without a high risk of contaminating the surroundings and the following patient samples. Recently other closed systems have been developed such as the LuciraCOVID-19 All-in-One single-use Test kit (https:// www. lucir aheal th. com/) and the single-use COVID-19 test from Visby medical (https:// www. visby medic al. com/) which are all-in-one single use NAAT tests that eliminates the need for opening the tube after amplification. In contrast to these single-use molecular test kits, the Egoo Health System can be used unlimited times and only requires replacing the Egoo assay capsule after use. Therefore, the Egoo Health System can be used for many subsequent assays, including tests for other respiratory viruses and biochemical markers such C-reactive protein (CRP) and Phenylalanine (PHE) (https:// www. egoo. health).
Due to limiting heating capacity of the small Egoo instrument, we developed an isothermal SARS-CoV-2 assay based on RT-SIBA 28,29 and SYBR green detection. The SARS-CoV-2 RT-SIBA assay is performed at 44 °C and can be used in both the Egoo instrument and in high-throughput format using standard PCR instruments. The gold standard RT-PCR test is dependent on the heating and cooling of the sample for the amplification reaction to occur. Heating and cooling require specialized equipment and so far, the most common PCR instruments have been too big and heavy to handle outside the laboratory. Recently, the CovidNudge portable RT-PCR platform 27 and the single-use RT-PCR device from Visby medical 26 were developed which opens the possibility to perform the gold standard RT-PCR outside the laboratory. The Egoo instrument uses similar fluorescence optics as a Table 3. The clinical sensitivity and specificity of the SARS-CoV-2 Egoo Health system compared to different RT-PCR platforms (n = 1154). NA, nucleic acids; P, positive; N, negative; TP, true positive; TN, true negative; FP, false positive; FN, false negative; SE, sensitivity; SP, specificity; CI, confidence interval; OP, oropharyngeal swab; PBS, phosphate-buffered saline, UTM, universal transport medium. a Diagnosed with direct lysis using SIBA lysis/reaction buffer and the RT-PCR for the E-gene 33  www.nature.com/scientificreports/ PCR instrument and as such we observe a similar performance of the SARS-CoV-2 RT-SIBA assay in the two instruments with an analytical sensitivity of 25 viral RNA copies per reaction. For methodological simplicity, we developed an extraction-free SARS-CoV-2 RT-SIBA assay that uses a specialized SIBA lysis/reaction buffer containing mild detergents. During the COVID-19 pandemic, NA extraction has proved not only to be time-consuming, and has caused bottlenecks due to lack/shortage of consumables. Therefore, many laboratories have been forced to look for alternative methods to NA extraction such as direct use of the crude sample using either heat or detergents for inactivation and lysis of the virus. This has proven to be almost as sensitive and specific as the gold standard purification methods [5][6][7]9,37 and after optimization, we ended with a simplified sampling workflow for the SARS-CoV-2 Egoo capsule that can be used with the Egoo instrument.
When performing the SARS-CoV-2 RT-SIBA assay in a PCR instrument a melting curve analysis can be performed to test the specificity of the assay. However, this is not possible in the Egoo instrument and therefore sample dilution must be performed to reduce non-target amplification, e.g., by adding a high sampling volume (10 ml) to the collection tube or diluting the sample 10-fold. Studies have shown that direct use of nasopharyngeal or oropharyngeal samples dissolved in PBS, Saline, and UTM without NA extraction can inhibit direct RT-PCR 9,37 and dilution of samples (or reducing the sample input volume into the RT-PCR reaction) reduced the inhibitory effect 37 . The 10-fold dilution of the sample before analysis in the Egoo instrument will therefore not only eliminate non-target amplification but may also eliminate inhibitors that might otherwise influence the SARS-CoV-2 RT-SIBA reaction. Another advantage of the dilution workflow is that the SARS-CoV-2 RT-SIBA assay is compatible with several different VTMs, UTMs and antigen buffer systems. We show that SARS-CoV-2 Egoo Health System is compatible with several of the widely used antigen buffers systems and therefore can be used as a confirmatory NAAT test of patients tested positive or negative with a rapid antigen test 4 .
The SARS-CoV-2 Egoo Health System showed a sensitivity between 87.0 and 94.7% dependent on the reference NAAT test used. We obtained the lowest sensitivity of 87.0% (100/115) when the assay was compared to the Cobas SARS-CoV-2 & Influenza A/B NAAT test on the Cobas Liat System. The main reason for this difference is the sample input volume into the system. For the Corbas Liat system 200 µl sample is loaded directly into the assay cartridge, whereas for the Egoo system 20 µl sample is 10-fold diluted before 20 µl of the diluted sample is loaded into the SARS-CoV-2 Egoo capsule, meaning that there is a 100-fold difference in sample input between the two systems. Compared to the Roche Flow/MGI-BGI RT-PCR reference method which include a NA purification, an up concentration (180-33 µl) of the RNA and detection of two targets, we obtained a sensitivity of 91.8% (482/525) with the SARS-CoV-2 Egoo Health System. For samples with ct-values below 35 the Egoo system achieved a sensitivity of 96.6% (450/466) meaning that samples with low viral load (high ct-values) could potentially not be detected with the Egoo Health System.
The clinical performance of the SARS-CoV-2 Egoo Health system is similar to the clinical performance of the COVID-19 test on the Cue Health Monitoring system 25 . The Cue Health Monitoring System (Cue Cartridge Reader) have recently developed a COVID-19 Test Cartridge which was evaluated on 292 symptomatic and asymptomatic patients 25 . They showed a positive percent agreement (PPA) of 91.7% (22/24) and a negative percent agreement (NPA) of 98.4% (239/243) compared to a reference NAAT test using standard nasopharyngeal swab 25 which is comparable to our sensitivity and specificity of 91.8% and 98.2% respectively when testing 1154 oropharyngeal swabs.
The Egoo Health System and SARS-CoV-2 RT-SIBA assay presented here has recently been CE-marked for professional use (Qlife), and we are currently trying to develop a multiplex probe based SARS-CoV-2 RT-SIBA assay containing the human RNaseP as an internal control 38,39 that can be used directly on anterior nasal swabs.