key: cord-0995372-nf30qlfi authors: Reijns, M. A.; Thompson, L.; Acosta, J. C.; Black, H. A.; Sanchez-Luque, F. J.; Diamond, A.; Daniels, A.; O'Shea, M.; Uggenti, C.; Sanchez, M. C.; McNab, M. L.; Adamowicz, M.; Friman, E. T.; Hurd, T.; Jarman, E. J.; Lee Mow Chee, F.; Rainger, J. K.; Walker, M.; Drake, C.; Longman, D.; Mordstein, C.; Warlow, S. J.; McKay, S.; Slater, L.; Ansari, M.; Tomlinson, I. P.; Moore, D.; Wilkinson, N.; Shepherd, J.; Templeton, K.; Johannessen, I.; Tait-Burkard, C.; Haas, J. G.; Gilbert, N.; Adams, I. R.; Jackson, A. P. title: A sensitive and affordable multiplex RT-qPCR assay for SARS-CoV-2 detection date: 2020-07-16 journal: nan DOI: 10.1101/2020.07.14.20154005 sha: 7b20ab95e844130487bf1d2bff207ccac7ad81b2 doc_id: 995372 cord_uid: nf30qlfi With the ongoing COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, there is need for sensitive, specific and affordable diagnostic tests to identify infected individuals, not all of whom are symptomatic. The most sensitive test involves the detection of viral RNA using RT-qPCR, with many commercial kits now available for this purpose. However, these are expensive and supply of such kits in sufficient numbers cannot always be guaranteed. We therefore developed a multiplex assay using well-established SARS-CoV-2 targets alongside internal controls that monitor sample quality and nucleic acid extraction efficiency. Here, we establish that this test performs as well as widely used commercial assays, but at substantially reduced cost. Furthermore, we demonstrate >1,000-fold variability in material routinely collected by nose-and-throat swabbing. The inclusion of a human control probe in our assay provides additional information that could help reduce false negative rates. The COVID-19 pandemic originated in Wuhan (China) in December 2019 and at the time of writing 14 has infected more than 13.1 million people worldwide, resulting in well over 0.57 million COVID-19-15 related deaths. In many countries the number of active cases is now declining, largely due to 16 increased public awareness and effective public health strategies centred on reducing the rate of 17 transmission. However, the number of cases worldwide is still on the increase with around 100,000 18 new cases recorded every day at the beginning of June and more than 200,000 new daily cases since 19 the beginning of July. Many of these new infections are now occurring in lower-middle-income 20 countries. Also, as lockdown measures are widely being eased there is an increased risk of a 21 renewed rise in infection rates, as evidenced by current trends observed in e.g. Iran and the USA. 22 Therefore, effective and affordable testing strategies to enable effective and widespread population 23 surveillance will continue to be important. The most sensitive test to diagnose infected individuals 24 involves the detection of SARS-CoV-2 viral RNA using RT-qPCR, most commonly using samples 25 collected using nasopharyngeal (nose-and-throat) swabs, although there is increasing evidence that 26 the use of saliva may be a valid alternative [1] . Many commercial kits are now available, most of 27 which employ multiplex RT-qPCR detecting 2 or 3 different SARS-CoV-2 targets, and generally 28 include an internal control to show successful nucleic acid extraction. However, such kits are often 29 costly and their supply in sufficient numbers cannot always be guaranteed. We therefore developed 30 a similar multiplex assay using well-established SARS-CoV-2 targets and internal controls, which can 31 be carried out at a significantly lower cost and provides more flexibility to ensure resilience against 32 potential shortages in reagent supplies. 33 assays were published on the WHO website [4] . Based on the data available at the time, we decided 38 to focus our initial efforts on targeting the following SARS-CoV-2 genes: E (envelope), RdRp (RNA-39 dependent RNA polymerase) and N (nucleocapsid) [5, 6] . proposed the E gene 40 as a useful target for first line screening, with the RdRp gene suggested as a good target for 41 confirmatory/discriminatory assays [7, 8] . The N gene was central to the USA Centers for Disease 42 Control and Prevention (CDC) in vitro diagnostics emergency protocol, with three different 43 primer/probe sets used against different portions of this viral gene [4] . The CDC protocol also 44 included a probe against human RPP30, a single copy gene encoding the protein subunit p30 of the 45 Ribonuclease (RNase) P particle, to ensure the presence of a sufficient number of cells in patient 46 samples and successful isolation of intact nucleic acids. 47 In early versions of these protocols, all probes were labelled with fluorescein amidite (FAM), and 48 separate reactions were therefore needed to detect each target. To increase efficiency, we 49 developed a multiplex assay using 4 different fluorescent labels (FAM, HEX, CAL Fluor Red 610 and 50 Quasar 670) for each of the probes, allowing their detection in a single reaction. In the final version 51 of our assay, we use previously described primers and probes against the well-established SARS-CoV-52 2 E and N gene targets, as well as two internal controls: human RPP30 and Phocine Herpes Virus 1 53 (PhHV-1, hereafter referred to as PhHV). The rationale behind the human cellular control is that a 54 considerable number of patients with clinical and radiological signs of COVID-19 are PCR negative; 55 and the poor quality of swab samples with no or little usable patient material is one possible 56 explanation for this [9] . In essence, the RPP30 control provides a measure of sample quality. In 57 If paired with an in-house RNA extraction protocol, our assay can be performed for less than £2 positive. In addition, there was one sample (P53) that was negative with TaqPath, but positive for 161 both N1E-RP and N2-ERP, albeit with very high Ct values (between 35.7 and 39.2), close to the limit 162 of detection. 163 In addition to the patient samples (n=108 total), 8 quality control samples from Quality Control for 164 Molecular Diagnostics (QCMD, an external quality assessment organisation) were also tested. All 165 assays (N1E-RP, N2E-RP and TaqPath) gave the same results for the QCMD control samples: 5 tested 166 positive and 3 negative. These results are as expected when compared to the sample identities and 167 data provided by QCMD (Table 1 , Fig S3) . Altogether, our data establish that the sensitivity of the 168 N1E-RP and N2E-RP assays is similar to that of the TaqPath assay (Fig 2) . 169 The human RPP30 control demonstrates substantial variability with >1,000-fold difference in 171 patient material in swabs 172 In contrast to consistent Ct values for the PhHV internal control, indicating reproducible nucleic acid 173 extraction, the range of Ct values for the human RPP30 control was much greater, consistent with 174 considerable variability in the amount of usable material present in different patient samples (Table 175 S4, Fig 3 and S1B) . Although, the RPP30 control worked for all samples, Ct values ranged from 20.3 176 to 31.7 for the N1E-RP assay and from 20.3 to 32.1 for the N2E-RP assay. This equates to a difference 177 of between 2,700 and 3,700-fold in extracted nucleic acids between the best and the worst samples. 178 This could mean that compared to a sample of high quality (lowest RPP30 Ct) with a theoretical 179 SARS-CoV-2 Ct value of 28.6, a sample from the same patient of low quality (highest RPP30 Ct) would 180 not be picked up as positive, assuming a SARS-CoV-2 detection limit of 40. The absence of a "sample 181 quality" control such as RPP30 therefore substantially increases the chance of false negative test 182 results when working with suboptimal samples. Complete absence of RPP30 signal (undetected or Ct 183 >40) clearly indicates that the test result cannot be interpreted and that a repeat test is therefore 184 required. However, utilising RPP30 Ct values when interpreting an apparent SARS-CoV-2 negative 185 9 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) Here, we describe a user-friendly protocol for an accurate and affordable SARS-CoV-2 RT-qPCR test. 198 We provide detailed materials and methods to enable others to rapidly set up this assay in their own 199 laboratory or to adapt it to locally available equipment and reagents. Our assays have high analytical 200 sensitivity, equivalent to commercial CE-IVD kits. Ultimately, the clinical sensitivity of any of these 201 diagnostic tests is influenced by multiple factors, including sample timing relative to symptom onset, 202 sample type and sample quality. The inclusion of a human control (RPP30) in our assays provides an 203 internal sample quality control that will aid interpretation of test results, and should contribute to 204 reducing false negative results. 205 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted July 16, 2020. . The methods below describe the materials and methods we employed in the development and 208 testing of our assays. There should be some flexibility in terms of the precise reagents and 209 instruments used to perform these multiplex RT-qPCR assays. For example, many companies are 210 able to synthesise high-quality primers and probes with different labels, and alternative Real-Time 211 PCR machines can be used, as long as they are able to detect different channels simultaneously and 212 have been calibrated. A different spike-in to our PhHV control could be used, e.g. lentiviral particles 213 with a GFP transgene and primers/probe targeting GFP would be one option. Also, it is likely that 214 further improvements can be made to our protocol, either generally or to match with local 215 requirements/capabilities. For example, the use of control primers/probe specific to a human RNA 216 transcript (the RPP30 primers/probe described here detect both RNA and genomic DNA) would give 217 even greater confidence in sample quality, i.e. ensuring that it contains intact RNA. However, it 218 should be stressed that any changes to the protocol may change the sensitivity of SARS-CoV-2 219 detection, and this should be checked using a thorough validation procedure (e.g. as described here) 220 before using these methods for diagnostic purposes. 221 222 Samples were collected from symptomatic individuals by trained healthcare professionals using 224 combined nose-and-throat swabbing, and processed for diagnostic testing using validated CE-IVD 225 assays. Excess samples were then used to validate the in-house multiplex assays, with specimen 226 anonymization by coding, compliant with Tissue Governance for the South East Scotland Scottish 227 Academic Health Sciences Collaboration Human Annotated BioResource (reference no. SR1452). A 228 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted July 16, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted July 16, 2020. . used as instructed by the manufacturer. Early tests using mono or duplex RT-qPCR assays were 273 performed using FAM-labelled probes or FAM and HEX-labelled probes respectively (sequences as in 274 Table 2 ). Ultimately, experiments using the 4-plex assay were performed as described below, with a 275 user-friendly protocol provided in Supplementary Material, Protocol 1. 276 13 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2020. . For the 4-plex assays, the same primer/probe sequences and concentrations were used as for the 277 mono/duplex assays. However, different labels and quenchers were used to allow simultaneous 278 detection of four different targets (Table 2) . Reaction master mixes were prepared (20 μl per 279 reaction) for each assay, before adding 5 μl of template RNA per reaction, brief centrifugation and 280 starting the PCR program. For the RdE-RP 4-plex assay, per reaction 12.5 μl of One-Step mix, 5.5 μl of 281 nuclease-free water, 2 μl of 12.5x primer/probe mix and 5 μl of template RNA were mixed. for RdRp [7, 8] , N1/N3 and N2 [4] . An equimolar mix of all RNAs was prepared at 2.5 x 10 8 copies/µl, 291 and aliquots stored at -80°C. Dilution series were prepared in nuclease-free water, in Eppendorf DNA 292 LoBind tubes (Cat. No. 10051232), at 2,000, 200, 20, 10, 2 and 0.2 copies/µl. 293 Quality Control for Molecular Diagnostics (Glasgow, UK) provided controls as part of the "QCMD 296 2020 Coronavirus Outbreak Preparedness (CVOP) EQA Pilot Scheme". After they were tested blind 297 using our assays, expected results along with sample identities were provided by QCMD. 298 299 14 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2020. . . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2020. . is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2020. 18 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2020. . Also, see Table S2 and S3. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2020. . Table S1 and S4. 20 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2020. . 2 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2020. . is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2020. . Log10 dPCR values were obtained by QCMD using a digital droplet PCR assay (modified from [7, 8] ). Also, see Table 1 . 4 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2020. . It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2020. . https://doi.org/10.1101/2020.07.14.20154005 doi: medRxiv preprint Table S2: Fig 1A and B 6 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2020. . Fig 1C) neat 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2020. . is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2020. . It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2020. . https://doi.org/10.1101/2020.07.14.20154005 doi: medRxiv preprint Supplement: An affordable multiplex RT-qPCR assay for SARS-CoV-2 detection July 14, 2020 Dissolve primers and probes (Table P1 ; HPLC purified) in 10 mM Tris, 0.1 mM EDTA, pH 8.0 (IDTE Cat. No. 11-05-01-09, or similar) for 100 μM stocks. Prepare primer/probe mixes (Tables P2-P7) , and store aliquots at -20°C. Working stocks can be stored at 4°C. TxRd_E_Sarbeco_P1* CFR-610-ACACTAGCCATCCTTACTGCGCTTCG-BHQ2 SARS-CoV-2, E gene [1, 2] 2019-nCoV_N1-F GACCCCAAAATCAGCGAAAT SARS-CoV-2, N gene [3] 2019-nCoV_N1-R TCTGGTTACTGCCAGTTGAATCTG SARS-CoV-2, N gene [3] 2019-nCoV_N1-P FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1 SARS-CoV-2, N gene [3] 2019-nCoV_N2-F TTACAAACATTGGCCGCAAA SARS-CoV-2, N gene [3] 2019-nCoV_N2-R GCGCGACATTCCGAAGAA SARS-CoV-2, N gene [3] 2019-nCoV_N2-P FAM-ACAATTTGCCCCCAGCGCTTCAG-BHQ1 SARS-CoV-2, N gene [3] Hs_RPP30-F AGATTTGGACCTGCGAGCG Human RPP30 [3] Hs_RPP30-R GAGCGGCTGTCTCCACAAGT Human RPP30 [3] HEX-Hs_RPP30-P HEX-TTCTGACCTGAAGGCTCTGCGCG-BHQ1 Human RPP30 [3] PhHV-F GGGCGAATCACAGATTGAATC PhHV-1, Glycoprotein B [4] PhHV-R** GCGGTTCCAAACGTACCA(A) PhHV-1, Glycoprotein B [4] Cy5-PhHV-P*** Quasar-670-TTTTTATGTGTCCGCCACCATCTGGATC-BHQ2 PhHV-1, Glycoprotein B [4] * Probe named TxRd for simplicity, CAL Flour Red (CFR-610) has virtually identical properties to TexRed ** Reverse primer GCGGTTCCAAACGTACCA used for our work; GCGGTTCCAAACGTACCAA used in [4] ; both should work equally *** Probe named Cy5 for simplicity. Quasar 670 has virtually identical properties to Cy5 12 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2020. . https://doi.org/10.1101/2020.07.14.20154005 doi: medRxiv preprint 13 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2020. . https://doi.org/10.1101/2020.07.14.20154005 doi: medRxiv preprint Add the viral spike-in control to the lysis buffer master mix before sample inactivation. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2020. . 1. Prepare an equimolar mix of all RNAs at 2.5 x 10 8 copies/µl. Store aliquots of this solution at -80°C. 2. Prepare 10 4 copies/µl positive RNA controls and store 5 µl aliquots at -80°C for single use per plate. Mix 5µl of 2.5 x 10 8 copies/µl with 620 µl of water to give 2 x 10 6 copies/µl. Dilute this 20 µl plus 180 µl water giving a 2 x 10 5 copies/µl solution, and 20 µl plus 380 µl giving 10 4 copies/µl. 3. For each plate, a 25 copies/µl solution is made by diluting the 10 4 copies/µl solution by mixing: 2 µl with 98 µl water, then 12.5 µl of this with 87.5 µl water Of this 25 copies/µl solution, 2 µl is added to well H12 along with 3 µl of water, to give the 50 copy positive control on each plate. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted July 16, 2020. . In principle, the buffers and solution below can replace those of the equivalent buffers in the Omega Mag-Bind Viral DNA/RNA 96 Kit (Cat. No. M6246). Lysis and wash buffers can either be replaced with guanidine thiocyanate (GnSCN) or guanidine hydrochloride (GnHCl) containing solutions, depending on reagent availability. In our preliminary tests, all performed equally well (data not shown). All lysis buffers (Omega TNA, GnSCN Lysis Buffer, GnHCl Lysis Buffer, each with/without isopropanol) were shown to inactivate coronavirus after 15 min incubation (Fig S4) . Briefly, to determine whether lysis buffers inactivate coronaviruses, 200 µl CoV 229E-GFP [5, 6] stock (9.6x10 5 pfu/ml in DMEM, 10% FCS, 1% NEAA) was mixed with lysis buffer at the recommended ratio (240 µl lysis buffer without isopropanol or 520 µl lysis buffer with isopropanol, i.e. 240 µl buffer and 280 µl isopropanol). For positive infection controls, virus was mixed with 240 or 520 µl medium. All mixes were inverted 8 times and incubated at room temperature for 15 min. Cytotoxic components were then removed by centrifugation at 4°C using Microcon filter columns (Millipore; 30 kDa cut-off), and two 0.5 ml PBS washes, similar to previously described methods [7, 8] . Remaining virus particles were then resuspended in 200 µl DMEM and 50 µl of a 1/100 dilution added to HUH7 cells (a cell line permissive to infection by CoV 229E). Cells were seeded the previous day at 1.8x10 4 /well in a black 96-well plate (Corning), and were at ~80% confluence for infection. Cultures were monitored daily for cell viability, cytopathic effects and GFP expression using microscopy. No significant cell death was observed for any of the samples. Relative fluorescence was measured using a Clariostar BMG Plate Reader at 72 h, with fluorescence for a non-infected control set to zero. No fluorescence was observed for any of the lysis buffer treated samples (Fig S4) , and fluorescence microscopy confirmed the absence of GFP positive cells (data not shown), consistent with complete viral inactivation. In our preliminary tests, we used the March 2020 version of the protocol provided with the Omega Mag-Bind Viral DNA/RNA 96 Kit to test viral nucleic acid isolation with our own solutions and reagents (see below). We used the Mag-Bind Particles CNR from the Omega kit, and although we have not yet tested this, we expect that these can be replaced by SeraSil-Mag silica beads (Cytiva, . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted July 16, 2020. . https://doi.org/10.1101/2020.07.14.20154005 doi: medRxiv preprint Supplement: An affordable multiplex RT-qPCR assay for SARS-CoV-2 detection July 14, 2020 A43074) and KingFisher 96 microplates (Cat. No. 97002540). Alternative robots could be used; and manual purifications are also possible. 18 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted July 16, 2020. . Saliva is more sensitive for SARS-CoV-2 detection in COVID-19 patients than nasopharyngeal swabs A Novel Coronavirus from Patients with Pneumonia in China Comparison of SARS-CoV2 N gene real-time RT-PCR targets and commercially available mastermixes Emerging SARS-CoV-2 mutation hot spots include a novel RNA-dependent-RNA polymerase variant Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China COVID-19 RT-qPCR protocol v2 Available from Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR Correlation of Chest CT and RT-PCR Testing in Coronavirus Disease 2019 (COVID-19) in China: A Report of 1014 Cases Quantitation of viral load using real-time amplification techniques Routine use of a highly automated and internally controlled real-time PCR assay for the diagnosis of herpes simplex and varicella-zoster virus infections Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT-qPCR primer-probe sets Protocol in brief (tested with home-made solutions; based on Omega Per sample, prepare 528 µl master mix: 240 µl lysis buffer, 8 µl carrier RNA (1 µg/µl) and 280 µl isopropanol 2. Add 200 µl of patient sample in VTM, mix thoroughly KingFisher Flex, loop though 3 times: 2 min fast mix, 30 s half mix, 30 s bottom mix 4. Employ magnetic separation (Collect beads Wash beads with 400 μL VHB wash buffer (Release beads, 3 min fast mix Wash beads with 500 μL SPR Wash buffer (Release beads, 2 min fast mix Wash beads with 500 μL SPR Wash buffer (Release beads, 2 min fast mix Air dry magnetic beads (10 min, above well) Elute nucleic acids in 50 µl nuclease-free water (Release beads, 5 min medium mix Protocol in brief (not tested with home-made solutions; based on Omega Per sample, mix 240 µl lysis buffer and 1 µl carrier RNA (1 µg/µl) Add 200 µl of patient sample in VTM, mix thoroughly; incubate for >15 min KingFisher Flex, loop though 3 times: 2 min fast mix, 30 s half mix, 30 s bottom mix 4. Employ magnetic separation (Collect beads Wash beads with 350 μL VHB wash buffer (Release beads, 3 min fast mix Wash beads with 350 μL SPR Wash buffer (Release beads, 2 min fast mix Wash beads with 350 μL SPR Wash buffer (Release beads, 2 min fast mix Air dry magnetic beads (10 min, above well) Elute nucleic acids in 50 µl nuclease-free water (Release beads, 5 min medium mix COVID-19 RT-qPCR protocol v2 Available from Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR Routine use of a highly automated and internally controlled real-time PCR assay for the diagnosis of herpes simplex and varicella-zoster virus infections Dendritic cell-specific antigen delivery by coronavirus vaccine vectors induces long-lasting protective antiviral and antitumor immunity Reverse genetics of coronaviruses using vaccinia virus vectors The effect of a nondenaturing detergent and a guanidinium-based inactivation agent on the viability of Ebola virus in mock clinical serum samples Evaluation of heating and chemical protocols for inactivating Supplement: An affordable multiplex RT-qPCR assay for SARS-CoV-2 detection July 14, 2020