key: cord-0742216-nrkdkir6 authors: Thom, Ruth E.; Eastaugh, Lin S.; O’Brien, Lyn M.; Ulaeto, David O.; Findlay, James S.; Smither, Sophie J.; Phelps, Amanda L.; Stapleton, Helen L.; Hamblin, Karleigh A.; Weller, Simon A. title: Evaluation of the SARS-CoV-2 inactivation efficacy associated with buffers from three kits used on high-throughput RNA extraction platforms date: 2021-04-15 journal: bioRxiv DOI: 10.1101/2021.04.14.439928 sha: 34fb6e1a1b72a962e681de1fc217d24ce9013c0a doc_id: 742216 cord_uid: nrkdkir6 Rapid and demonstrable inactivation of SARS-CoV-2 is crucial to ensure operator safety during high-throughput testing of clinical samples. The inactivation efficacy of SARS-CoV-2 was evaluated using commercially available lysis buffers from three viral RNA extraction kits used on two high-throughput (96-well) RNA extraction platforms (Qiagen QiaCube HT and the ThermoFisher Kingfisher Flex) in combination with thermal treatment. Buffer volumes and sample ratios were chosen for their optimised suitability for RNA extraction rather than inactivation efficacy and tested against a representative sample type; SARS-CoV-2 spiked into viral transport medium (VTM). A lysis buffer from the MagMax Pathogen RNA/DNA kit (ThermoFisher), used on the Kingfisher Flex, which included guanidinium isothiocycnate (GITC), a detergent, and isopropanol demonstrated a minimum inactivation efficacy of 1 x 105 TCID50/ml. An alternative lysis buffer from the MagMax Viral/Pathogen Nucleic Acid kit (Thermofisher) also used on the Kingfisher Flex and the lysis buffer from QIAamp 96 Virus QIAcube HT Kit (Qiagen) used on the QiaCube HT (both of which contained GITC and a detergent) reduced titres by 1 x 104 TCID50/ml but did not completely inactivate the virus. Heat treatment alone (15 minutes, 68 °C) did not completely inactivate the virus, demonstrating a reduction of 1 x 103 TCID50/ml. When inactivation methods included both heat treatment and addition of lysis buffer, all methods were shown to completely inactivate SARS-CoV-2 inactivation against the viral titres tested. Results are discussed in the context of the operation of a high-throughput diagnostic laboratory. (ThermoFisher), used on the Kingfisher Flex, which included guanidinium isothiocycnate 23 (GITC), a detergent, and isopropanol demonstrated a minimum inactivation efficacy of 1 x 24 10 5 TCID 50 /ml. An alternative lysis buffer from the MagMax Viral/Pathogen Nucleic Acid kit 25 (Thermofisher) also used on the Kingfisher Flex and the lysis buffer from QIAamp 96 Virus 26 QIAcube HT Kit (Qiagen) used on the QiaCube HT (both of which contained GITC and a 27 detergent) reduced titres by 1 x 10 4 TCID 50 /ml but did not completely inactivate the virus. 28 Heat treatment alone (15 minutes, 68 °C) did not completely inactivate the virus, 29 demonstrating a reduction of 1 x 10 3 TCID 50 /ml. When inactivation methods included both 30 heat treatment and addition of lysis buffer, all methods were shown to completely 31 inactivate SARS-CoV-2 inactivation against the viral titres tested. Results are discussed in 32 the context of the operation of a high-throughput diagnostic laboratory. 33 Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) belongs to the 35 Coronaviridae family and is the causative agent of the respiratory illness, coronavirus 36 disease (1). The enveloped positive-sense single-stranded RNA virus was first 37 discovered in early 2020 after a cluster of viral pneumonia cases of unknown cause were 38 reported in the Hubei Province of China (2). The virus is highly contagious in humans and in 39 March 2020 The World Health Organisation (WHO) declared a global pandemic (3). 40 Diagnostic testing is critical in the fight against the COVID-19 pandemic (4), not just for 41 patients displaying symptoms but also for asymptomatic carriers and pre-symptomatic safety cabinet. 48 Real-time reverse transcriptase polymerase chain reaction (RT-PCR) is the gold standard test 49 to for the detection of SARS-CoV-2 from nasopharyngeal swab samples (8). Inactivation of 50 viral pathogens prior to PCR is typically carried out at the same time as extraction of viral 51 nucleic acids from samples, with chemical or physical methods employed. Typically buffers 52 provided in nucleic acid extraction kits contain chaotropic salts, solvents, and detergents to 53 lyse the virus. Guanidinium salts, such as guanidinium thiocyanate (GITC), are chaotropic 54 agents found in many lysis buffers which in some cases have been demonstrated to 55 inactivate viral pathogens, including alphaviruses, flaviviruses, filoviruses and a bunyavirus 56 (9, 10). Other reports though suggest that a combination of a GITC containing extraction 57 buffer (such as Qiagen AVL) and a solvent (such as ethanol), is required for the inactivation 58 of viruses such as Ebola virus (11) The inactivation efficacy of the lysis buffers in all three protocols was evaluated with and 108 without the inclusion of a heat step. To quantify and determine the viability of the virus following inactivation, the samples were 135 prepared for TCID 50 end-point dilution assay (20) and the remaining sample underwent 136 three rounds of serial passage in tissue culture flasks. 137 In brief, TCID 50 assay was performed using Vero C1008 cells prepared in 96-well microtitre 138 plates to achieve confluent monolayers on the day of assay. To all wells of column 1 of the 139 plate 100 µl of test sample was added. From column 1, 20 µl of sample was transferred 140 sequentially across the plate to achieve a 10-fold serial dilution to column 9. Cells in 141 columns 11 and 12 were left in TCM as controls. Plates were incubated in a humidified 142 atmosphere for 3 -4 days at 37 °C, after which they were scored for cytopathic effects (CPE) 143 by microscopic observation. The TCID 50 value was calculated by the method of Reed and 144 Muench (21). 145 For secondary confirmation of viral inactivation, all of the remaining sample (approx. 180 146 µl) was added to confluent monolayer of Vero C1008 cells in a 12.5 cm 2 tissue culture flask. 147 Flasks were incubated in a humidified atmosphere for 3 -4 days after which presence or 148 absence of cytopathic effect was recorded. A total of three passages were performed and 149 CPE recorded after each round. To control for cross-contamination a set of un-infected 150 flasks were also prepared and supernatant passaged in parallel to the experimental samples. All data were graphically represented and statistically analysed using GraphPad Prism 8. 157 Kruskal-Wallis analysis of variance (ANOVA) was performed on data sets with Dunn's 158 multiple comparison post hoc. The inactivation of SARS-CoV-2 was assessed using three different RNA lysis buffers with and 161 without the inclusion of a heat step. The viability of virus was determined quantitatively 162 using the TCID 50 assay and qualitatively by serially passaging samples in flask. When virus was added to the Qiagen lysis buffer there was a statistically significant 5-Log 10 174 drop in virus titre from 4.4x10 5 TCID 50 /ml to below the lower limit of quantification (LLoQ) 175 (p=0.002) Complete inactivation was not achieved however, as virus was detected below 176 the LLoQ but this was not quantifiable. However by extrapolation it was estimated that the 177 titre was 6.2 TCID 50 /ml ( Figure 1A ). To confirm findings by TCID 50 assay viral samples were propagated in cell culture flasks over 199 a total of three passages to identify potential viral break-through. 202 and on average the limit of detection was 1.3 TCID 50 /ml. 203 When virus was added to TCM, CPE was present in all flasks as expected (Table 2 row 6, 204 positive control). No cell toxicity was observed from negative control samples were only was added to lysis buffer and washed as described previously (Table 2 row (14) or residual toxicity leading to reduced 248 sensitivity of the read-out of the assays (28). The wash steps employed here eliminated all 249 residual toxicity, allowing the sensitivity of our assay read-outs to be unaffected. 250 In our study, the chemicals used to assess the inactivation of SARS-CoV-2 were 251 combinations of GITC, detergent and solvent. The Qiagen protocol (using reagents from the 252 QIAamp 96 Virus QIAcube HT Kit) and the MagMax Protocol 2 (using reagents from the 253 MagMax viral/pathogen nucleic acid isolation kit) both included GITC and a detergent, (SDS 254 or zwittergent, respectively) ( Table 1) within the lysis buffer mix there were, therefore, three components likely to exert a 286 disruptive effect on the SARS-CoV-2 viral envelope. The reagent to sample ratio of 3.8 : 1 287 was also higher, with more than double the volume of lysis buffer mix added to each 288 sample, compared to the other two methods assessed (Table 1) . 289 Our results suggest that both a high reagent to sample ratio and the incorporation of a The use of heat to inactivate virus has been reported to reduce viral RNA stability (29, 30) 317 and depending on the target gene used for RT-PCR, incubation at 65 °C for 30 minutes can 318 significantly reduce the target copy numbers leading to false negative results of clinical 319 samples (18, 30) . The DCL has an accredited SARS-CoV-2 diagnostic workflow (31) 2. SARS-CoV-2 10 -4 dilution 3/3 1.7 x 10 3 * 3/3 5.9 x 10 2 * 3/3 3.0 x 10 2 * 3. SARS-CoV-2 10 -5 dilution 3/3 1.7 x 10 2 * 3/3 59.4 * 2/3 20.0 * 4. SARS-CoV-2 10 -6 dilution 3/3 17 * 1/3 2.0 * 1/3 0.7 * 5. SARS-CoV-2 10 -7 dilution 2/3 1.1 * 0/3 0 * 0/3 0 * Dstl ©Crown Copyright, 2021 25 6. SARS-CoV-2 + TCM 9/9 4.4x10 5 (3.8x10 5 ) 9/9 2.0x10 5 (2.3x10 5 ) 9/9 7.7x10 4 (4.8x10 4 ) 7. SARS-CoV-2 + lysis buffer 3/9