The study is not a medical health science research project but a method comparison study using surplus material from routine oropharyngeal samples collected at Nordsjællands 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 Nordsjællands 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 x H107 x D94 mm), and weight (470 g/1.04 lbs). It consists of an integrated optical microelectromechanical system (optical MOEM) 30 and has a dual optical unit for simultaneously measuring fluorescence and absorbance (Table 1). 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. The SARS-CoV-2 RT-SIBA Egoo capsule (W24 x H28 x D36 mm) (Table 2) consists of 140 µl frozen SARS-CoV-2 RT-SIBA Mastermix. The SARS-CoV-2 RT-SIBA Egoo capsules are stored at -20°C until their intended use.
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 minutes. 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.
Sample preparations and dilutions
Oropharyngeal swabs collected with flocked swabs (Jiangsu Hanheng Medical Technology, Copan) from patients were dissolved directly into either 1 ml SIBA lysis/reaction buffer (Aidian/Qlife), 1 ml PBS (Gibco), 1 ml Universal Transport Media (UTM) (Copan), 1 ml Virus Transport Media (VTM) (NEST Biotechnology) or 1 ml VTM (Mole Bioscience). To release virus from the flocked swabs and the sample collection tubes were incubated for 10 minutes at room temperature. Samples dissolved in PBS, UTM, VTM or antigen buffers (GenSure™ COVID-19 Antigen Rapid Test, ACRO Rapid Test, BIOSYNEX®COVID-19 Ag BSS, COVID-19 Ag (SD Biosensor)) were diluted 10-fold in SIBA lysis/reaction buffer (containing mild detergents and 80 mM Mg-acetate) before being applied to the SARS-CoV-2 RT-SIBA Mastermix (Qlife). Samples undergoing SARS-CoV-2 testing by RT-PCR on the BGI system at Hvidovre Hospital or the Corbas Liat System at Nordsjællands Hospital were used without further preparation. Purification of total NA in the BGI system was done on a MGISP-960 using the MGIEasy Magnetic Beads Virus DNA/RNA Extraction Kit (MGI Tech Co., Ltd.). The sample extraction amount was 180 µl and total NA was eluted in 33 µl.
The SARS-CoV-2 RT-SIBA Mastermix (Qlife) consists of mixing three reagents according to the following protocol. The RT-SIBA Mix A, Mix B and Oligomix were thawed on ice and mixed by vortexing. Mix A can in some instances form precipitates which can be re-dissolved by heating to 37–41°C followed by vortexing. For reactions performed in a PCR instrument the mastermix was prepared by mixing 7 µl Mix A, 7 µl Mix B, and 3.5 µl Oligomix per reaction. Mastermix (17.5 µl) was added to PCR tubes and 2.5 µl of sample (diluted 10-fold in SIBA lysis/reaction buffer) was added to the mastermix. The RT-SIBA reactions were performed using either the MX3005P (Stratagene) or CFX96 (BioRAD) PCR instrument. Fluorescence measurements were recorded every minute for 30 minutes at 44°C, followed by a melt curve analysis: 44°C − 95°C. For reactions performed in the Egoo device, Egoo capsules (Qlife) containing 140 µl of premade mastermix were thawed and loaded into the Egoo device after applying 20 µl of the sample (diluted 10-fold in SIBA lysis/reaction buffer). Before the reaction starts, the Egoo capsule is closed in the Egoo instrument once the piston mechanism seals the capsule with the plunger. Once the plunger has sealed the capsule tight, the Egoo instrument heats the capsule to 44°C for 30 minutes. During the reaction, the reagents within the capsule are mixed by vortexing (1000 rpm) for 3 seconds every 5 minutes. Fluorescence measurements are recorded 3 times every minute.
The E-gene assay from Charité Berlin 31 was used with the Luna® Universal Probe One-Step RT-qPCR Kit (NEB). Briefly described 12.5µl Luna Universal One-Step Reaction Mix, 1.25µl Luna WarmStart® RT Enzyme Mix, 0.5µl E_Sarbeco_F1 (20 µM), 0.5µl E_Sarbeco_R2 (20 µM), 0.25µl E_Sarbeco_P1 (20 µM), 7.5µl nuclease-free water and 2.5µl sample (purified RNA or sample diluted 10-fold in SIBA lysis/reaction buffer) were mixed and run with the following program: 10min at 55°C, 3min at 95°C, 45 cycles of 15 sec. at 95°C and 30 sec. at 58°C. The RT-PCR reactions were performed using either the MX3005P (Strategene), CFX96 (BioRAD), or AriaMx (Agilent), and fluorescence were captured using the FAM channel. At Nordsjællands hospital the Cobas SARS-CoV-2 & Influenza A/B NAAT test on the Cobas Liat System (Roche) was used directly (200 µl) in the cartridge according to the manufactures instructions. At Hvidovre Hospital a newly laboratory developed test adopted from32 targeting the E-gene and the N2-gene in SARS-CoV-2 and RNaseP as human target was used for RT-PCR analysis in the BGI system. Reactions were set up in a 20 µl reaction volume using 8 µL sample (purified RNA), 10 µl KiCqStart One-Step Probe RT-qPCR ReadyMix (Sigma-Aldrich), 1 µl 4 mM dUTP (0.2 mM in total) and 1 µl mix of primers and probes. Final concentrations of primers and probes were 500 nM CoV_E_F primer (5’- ACAGGTACGTTAATAGTTAATAGCGT-3’), 400 nM CoV_E_R primer (5’- ATATTGCAGCAGTACGCACACA-3’), 150 nM CoV_E_P probe (5’-LC610- ACACTAGCCATCCTTACTGCGCTTCG-BBQ-3’), 400 nM CoV_N2_F primer (5’- TTACAAACATTGGCCGCAAA-3’), 400 nM CoV_N2_R1 primer (5’- AAGGTGTGACTTCCATGCCA-3’), 150 nM CoV_N2_P FAM probe (5’-FAM- ACAATTTGCCCCCAGCGCTTCAG-BBQ-3’), 100 nM RNaseP_F primer (5’- AGATTTGGACCTGCGAGCG-3’), 100 nM RNaseP_R primer (5’- GAGCGGCTGTCTCCACAAGT-3’) and 125 nM RNaseP_P_Cy5 probe (5’-Cy5- TTCTGACCTGAAGGCTCTGCGCG-BBQ-3’). RT-PCR was performed on the LineGene 9600 instrument with the following PCR profile: 10 min of 50 ⁰C and 60 s of 95 ⁰C followed by 45 cycles of 95 ⁰C for 5 s and 60 ⁰C for 30 s.
Virus culture and RNA purification
Inactivated virus cultures for Epstein-Barr Virus (EBV)(B95-8), Parainfluenza virus type 1 (PIV-1), Adenovirus type 5 (Adv5), Respiratory Syncytial virus type A (RSV-A)(2006), Influenza A (H1N1pdm), (INFL A)(NY/02/07), Influenza A (H3N2), Influenza B (INFL B)(Yamagata/16/88), Rhinovirus A16, Enterovirus type 68 (EV-68)(2007), Human metapneumovirus (hMPV) (Peru2-2002), Coronavirus OC43, Coronavirs NL63, Coronavirus 229E, SARS-COV-2 (Italy-INMI1)(1.02 x 108 TCID50/mL), SARS-COV-2 (USA-WA1/2020)(3.09 x 108 TCID50/ml), SARS-COV-2 (Hong Kong/VM2000i06i/2020)(1.15 x 107 TCID50/mL) purchased from Helvetica Health Care were used directly by spiking into an oropharyngeal swab background resulting in a 10-fold dilution of the virus. QCMD panels for MERS (2019), RSV (2019), hMPV (2019) and coronavirus (2019) were purified using the MagNA Pure 96 system (Roche) and the DNA and Viral NA Small Volume Kit (Roche). The human SARS-CoV-2 isolate 2019-nCoV Munchen 1–2 2020/984 (026V-03883, EVAg) was cultured in VERO E6 cells, and the virus titre of the supernatant was determined to 1.6 x 107 TCID50/ml. In addition, the harvested supernatant was quantified to 1.2 x 107 RNA copies/ml using MagNA Pure purified RNA and a standard curve based on the synthetic SARS-CoV-2 RNA control (MT007544.1, Twist Bioscience) spiked into RNA from a SARS-CoV-2 negative oropharyngeal swab. The quantification was performed using the RT-PCR E-gene assay 31.
Retrospective SARS-CoV-2 positive and negative oropharyngeal patient samples were used to analyse the clinical sensitivity of the SARS-CoV-2 RT-SIBA assay using the Egoo instrument. 227 oropharyngeal swabs dissolved in PBS and diagnosed positive or negative for SARS-CoV-2 using direct lysis and the E-gene RT-PCR assay 31 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 RT-SIBA assay on the Egoo instrument. At Nordsjællands 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 hours using the SARS-CoV-2 RT-SIBA assay 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.