key: cord-0802472-yhstsznw authors: Kalnina, Lelde; Mateu-Regué, Àngels; Oerum, Stephanie; Hald, Annemette; Gerstoft, Jan; Oerum, Henrik; Nielsen, Finn Cilius; Iversen, Astrid K.N. title: A simple, safe and sensitive method for SARS-CoV-2 inactivation and RNA extraction for RT-qPCR date: 2020-06-30 journal: bioRxiv DOI: 10.1101/2020.06.29.179176 sha: b7101a9735ffaed217fffa33151c3c2ae40dd39d doc_id: 802472 cord_uid: yhstsznw The SARS-CoV-2 pandemic has created an urgent need for large amounts of diagnostic tests to detect viral RNA, which commercial suppliers are increasingly unable to deliver. In addition to the lack of availability, the current methods do not always fully inactivate the virus. Together, this calls for the development of safer methods for extraction and detection of viral RNA from patient samples that utilise readily available reagents and equipment present in most standard laboratories. We present a rapid and straightforward RNA extraction protocol for inactivating the SARS-CoV-2 virus that uses standard lab reagents. This protocol expands analysis capacity as the inactivated samples can be used in RT-qPCR detection tests at laboratories not otherwise classified for viral work. The method circumvents the need for commercial RNA purification kits, takes about 30 minutes from swab to PCR-ready viral RNA, and enables downstream detection of SARS-CoV-2 by RT-qPCR with very high sensitivity (~4 viral RNA copies per RT-qPCR). In summary, we present a rapid, safe and sensitive method for high-throughput detection of SARS-CoV-2, that can be conducted in any laboratory equipped with a qPCR machine. In mid-December 2019, reports emerged that patients in Wuhan, Hubei province, China, were 39 suffering from atypical pneumonia, and by start-January, the causative agent, severe acute 40 respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified (1, 2). The disease was named 41 coronavirus disease 2019 . The initial epicentre of virus spread seems to have been 42 the Huanan seafood wholesale market in Wuhan. Although the SARS-CoV-2 genome is very 43 similar to bat SARS-CoV-like coronaviruses (~96%), it carries unique sequence motifs in the 44 receptor-binding domain (RBD) of the Spike protein that binds to the human angiotensin-45 converting enzyme 2 (ACE2) receptor (3). These differences suggest that natural selection in an 46 intermediate host species optimised binding of SARS-CoV-2 to ACE2, and facilitated transmission 47 to, and spread between, humans. By mid-January 2020, the virus was found in Thailand and Japan 48 following which it spread worldwide (4, 5). As of June 2020, the US had the largest number of 49 identified SARS-CoV-2 infected individuals, but also several European countries, e.g., Italy, Spain, 50 United Kingdom, and France have large numbers of COVID-19 patients (6). As of today, cases of 51 COVID-19 are rapidly increasing in India, Mexico and parts of Africa and South America. 52 The possibility to rapidly test large numbers of individuals for the presence of SARS-CoV-2 is a 54 vital component in containing viral spread, in understanding the infectious fatality rate, and in 55 subsequently guiding the controlled reopening of our societies. In medical laboratories, the 56 presence of SARS-CoV-2 is commonly detected in a two-step process, where each step requires 57 different kits. Step one is the RNA extraction from patient swabs usually performed using a kit 58 from Qiagen or Roche (7), and step two is the detection of SARS-CoV-2, often achieved by a 59 reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR). With the rapidly growing 60 need for SARS-CoV-2 tests, commercial supplies are increasingly falling short on kits for both 61 6 To test the effect of GITC on the quality of both one-step and two-step RT-qPCR reactions, we 109 created dilution series of the OP and NP samples that allowed the final dilution in the subsequent 110 RT-reaction of the RT-qPCR to range from 8x to 100x (Figure 1) . For example, to achieve a 100x 111 final dilution in the RT-reaction of a 25 µL one-step RT-qPCR, the 10 µL sample was mixed with 112 190 µL RNase-free water to a dilution of 20x, followed by 5 µL of this sample mixed in a 25 µL 113 one-step RT-qPCR to a final dilution of 100x. (2 min at 50C, 2 min at 95C, followed by 40 cycles of 95C for 15 sec; 60C for 1 min). Finally, a 133 melting curve was recorded for 15 sec. at 95°C, 1 min at 60°C and 15 sec at 95°C. Data were 134 analysed using QuantStudio TM 12K Flex Software. To confirm that only mRNA was amplified, the 135 reactions were analysed by gel electrophoresis (data not shown). The B2M qPCR reaction proceeds 136 with a forward primer placed in exon 2 and a reverse primer spanning the exon 3/4 junction to 137 avoid amplification of the genomic B2M gene. With B2M cDNA, the primers produce an amplicon 138 of 97 nucleotides, whereas the amplicon from the B2M gene itself spans 1974 nucleotides. 139 140 The effect of GITC on a corresponding one-step SARS-CoV-2 RT-qPCR reaction was examined 142 using the dilution series of extracted RNA from OP and NP samples from the COVID-19 patient. An OP sample swab was collected from a healthy individual and processed as described above. To assess the sensitivity of the COVID-19 diagnostic test flow, the one-step RT-qPCR was 217 performed on a dilution series of a synthetic SARS-CoV-2 control RNA. RNA from an OP swab 218 from a healthy individual was extracted using the simplified GPC-extraction method, after which 219 some of the aqueous phase was spiked with the SARS-CoV-2 synthetic RNA to a final 220 concentration of 250000 copies/µL. This spiked sample was used to create a dilution series ranging 221 from 15625 to 3.8 copies/µL using the un-spiked aqueous phase from the sample as the diluent to 222 retain consistent amounts of GITC and swab components in the RT-qPCRs. In accordance with the 223 COVID-19 diagnostic test flow, each spiked sample was used at a final GITC dilution of 100x and 224 SARS-CoV-2 RNA was detected with N1 or N2 primers, and using RNase P primers as positive 225 control. 226 227 Amplicons were detected in all dilutions of the SARS-CoV-2 synthetic RNA down to 3.8 copies 228 with the N1 primers (Figure 2b) . This sensitivity is consistent with that reported for other primers 229 that target the N gene (N-Sarbeco, Tib-Molbiol, Berlin, Germany) that showed a detection limit of 230 8.3 copies/reaction when used with a commercial RNA extraction kit (MagNA Pure 96 system, 231 Roche, Penzberg, Germany) and the same one-step RT-qPCR (11). The ability to detect ~4 copies 232 of viral RNA in the RT-qPCR reaction translates into ~10 4 viral copies per swab, which is more 233 than 10 fold lower than the average virus load per NP or OP swab from symptoms onset to day five 234 (6.76x10 5 copies per swab) and later (3.44x10 5 copies per swab) (10, 11). The N2 primers proved 235 less sensitive, detecting synthetic RNA down to only 244 copies/reaction in our set-up. It cannot, 236 however, be excluded that the sensitivity is nearer the next testing point of 61 copies/reaction. The 237 negative control with no added virus showed no amplification, whereas efficient amplification was 238 observed with all positive control reactions targeting RNase P. Together, these data demonstrate 239 that the simplified GPC-extraction method allows for similar detection sensitivity in the one-step 240 RT-qPCR as a currently utilised kit-based RNA extraction methods. 241 242 Based on combined experiments, we outlined a COVID-19 diagnostic test flow from patient-to-243 result that covers patient sampling, RNA extraction by the simplified GPC-extraction method, and 244 one-step RT-qPCR detection (Figure 3) . prompting us to speculate that the RNA/GITC aqueous phase could be used directly in RT-qPCR 265 without precipitation, centrifugation, washing and solvation if the solution was diluted below the 266 ~75mM threshold. Our results robustly demonstrate that a dilution of 50x and 100x of the aqueous 267 phase is compatible with one-and two-step RT-qPCRs, respectively, and that approximately ~4 268 copies of SARS-CoV-2 can be detected, equivalent to ~10 4 virus copies per NP or OP swab. The 269 13 difference between the one-and two-step RT-qPCRs results are likely due to the extended period 270 of exposure of the PCR polymerase to GITC salts during the latter procedure. A new coronavirus associated with human respiratory disease in China Shi 325 ZL. 2020. 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