key: cord-0727701-eb6qw116 authors: Liu, G.; Jia, J.; Zhong, J.; Jiang, H.; Yang, Y.; Lu, X.; He, Z.; Zhu, Q. title: Ammonium Sulfate Addition Reduces the Need for Guanidinium Isothiocyanate in the Denaturing Transport Medium Used for SARS-COV-2 RNA Detection date: 2022-03-02 journal: nan DOI: 10.1101/2022.02.28.22271591 sha: 6a3444f5f25c9a478a36c7b61eaf6a61d1fc2836 doc_id: 727701 cord_uid: eb6qw116 Rapid identification of SARS-CoV-2 infected individuals through viral RNA detection followed by effective personal isolation remains the most effective way to prevent the spread of this virus. Large-scale RNA detection involves mass specimen collection and transportation. For biosafety reasons, denaturing viral transport medium has been extensively used during the pandemic. But the high concentrations of guanidinium isothiocyanate (GITC) in such media have raised issues around sufficient GITC supply and laboratory safety. Here, we tested whether supplementing media containing low concentrations of GITC with ammonium sulfate (AS) would affect the throat-swab detection of SARS-CoV-2 pseudovirus or a viral inactivation assay targeting both enveloped and non-enveloped viruses. Adding AS to the denaturing transport media reduced the need for high levels of GITC and improved SARS-COV-2 RNA detection without compromising virus inactivation. Introduction 33 The ongoing COVID-19 pandemic has resulted in more than 422 million infections and 5.8 million 34 deaths worldwide since its emergence in late 2019 [1, 2] . It appears to have now entered a new wave 35 of infection sparking by the heavily mutated Omicron variant [3] . Although several vaccines (e.g., 36 mRNA vaccines, inactivated vaccines, and viral vector vaccines) have been conditionally approved 37 in some countries [4, 5] and various antiviral agents are in various stages of clinical trial [6, 7] , there 38 is currently no effective way to control the COVID-19 pandemic. Before all susceptible populations 39 are fully protected, it remains extremely important to deal with the pandemic by controlling the 40 infection sources and blocking the transmission routes, both of which rely on rapidly 41 identifying SARS-CoV-2 infected individuals and isolating them from the population. Nucleic acid-42 based methods are the fastest methods for detecting SARS-CoV-2, especially in the early infection 43 stages [8] . Such methods have played irreplaceable roles in the mass screening for SARS-CoV-44 2 infection. Large-scale nucleic acid testing has been implemented following local case reports where 45 possible community transmission is indicated and this approach has supported China's sustained 46 containment of . Wuhan (Hubei Province) performed city-wide mass screening using 47 reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) testing for nearly 10 million 48 people over a 10-day period [10] , and Qingdao (Shandong Province) tested 10.9 million people for 49 SARS-CoV-2 RNA in 5 days [11] . 50 Large-scale nucleic acid testing involves large-scale specimen collection and transportation. 51 Nasopharyngeal swabs and throat swabs are usually used in the specimen collection for SARS-CoV-52 2 RT-PCR testing [8] . After collection, specimens are generally preserved and transported in 53 nondenaturing or denaturing media. Nondenaturing media, such as conventional viral transport 54 medium and Amies transport medium, mainly contain saline buffer and antibiotics [12] [13] [14] . 55 Nondenaturing media are used to maintain the integrity and infectivity of the viruses in the specimen. 56 Therefore, nondenaturing media are used to detect viral nucleic acids and antigens or for culture-57 based viral detection. However, laboratory personnel risk infection when handling infectious 58 specimens during the testing process, especially during large-scale testing [15] . Denaturing transport 59 media usually contain guanidinium isothiocyanate (GITC) or other virus-inactivating denaturants, 60 which is why they are only suitable for nucleic acid detection. One advantage of denaturing transport 61 medium is its potential to reduce the risk of infection of laboratory personnel and improve SARS-62 CoV - acid testing, a GITC supply shortage occurred and its price doubled in early 2021 in China. 72 Therefore, optimizing the formulation by reducing the GITC content may be a way to help meet the 73 demand for SARS-CoV-2 nucleic acid screening and reduce the testing costs. Ammonium sulfate 74 (AS) is a low-cost inorganic salt with high water solubility from its ionic nature. It is often used for 75 protein precipitation and purification in the laboratory. High-concentration AS is also used to 76 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 March 2, 2022. ; https://doi.org/10.1101/2022.02.28.22271591 doi: medRxiv preprint enhance RNA stability in tissue samples [23, 24] . Here, we report that reducing the amount of GITC 77 in denaturing viral transport media while adding AS to them can improve the sensitivity of SARS-78 CoV-2 RNA detection without affecting the virus inactivation effect. 79 Materials and methods 80 Vero cells (ATCC, CCL-81) were cultured in Dulbecco's modified Eagle's medium (Gibco, NY, 82 USA) supplemented with 10% (v/v) fetal calf serum. SARS-CoV-2 pseudovirus (FNV-2019-nCoV-83 abEN, >10 8 copies/mL) was purchased from Fubio Biological Technology Co., Ltd. (Hangzhou, 84 China). Herpes simplex virus type 1 (HSV-1, VR-733) originated from the American Type Culture 85 Collection. Enterovirus 71 (EV71, C4 strain) was kindly provided by Dr Tao Peng, Guangzhou 86 Medical University, China. Both HSV-1 and EV71 were propagated and titred in Vero cells. SARS-87 CoV-2 pseudovirus was used directly. 88 Modified Primestore MTM (Molecular Transport Medium), which we used as a reference medium, 90 contains 3M GITC, 25 mM sodium citrate, 0.5% SLS (sodium lauryl sulfate) and 20 mM 91 ethylenediaminetetraacetic acid. Basing on the modified Primestore MTM, we prepared the 92 denaturing transport medium with various GITC concentrations and/or AS as indicated. 93 A throat swab sample from a healthy person was collected in 4 mL saline, and the 100 μ L of SARS-95 CoV-2 pseudovirus (>10 7 copies) we added to the sample was mixed in by vortexing. and finally used in a plaque reduction assay using previously described procedures [25, 26] . 114 Results and discussion 115 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 March 2, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 To evaluate the effect of adding AS to denaturing transport media on SARS-CoV-2 RNA detection, a 117 modified commercial denaturing transport medium containing 3M GITC was used as the basic 118 formula and reference reagent in the experiments. To mimic the sampling of SARS-CoV-2, a throat 119 swab sample from a healthy person was mixed with SARS-CoV-2 pseudovirus. It was then placed 120 into denaturing media containing different concentrations of GITC and AS and incubated at room 121 temperature or 37°C for 24 h, 72 h, 120 h or 5 d as indicated. After RNA extraction, qRT-PCR on the 122 SARS-CoV-2 ORF1ab and N genes was performed and comparatively analysed. 123 The results showed that reducing the GITC concentrations increased the Ct values, but adding 124 1M AS to the transport media reduced the Ct values of the ORF1ab gene and the N gene irrespective 125 of the 24-h, 72-h or 120-h incubation period ( Fig. 1A and 1B) . A further assay on the samples 126 incubated at RT or 37°C for 5 days revealed that adding 1M AS to transport media containing lower 127 GITC concentrations significantly reduced the Ct values when compared with PBS or the transport 128 medium containing 3M GITC (p<0.01) (Fig. 1C) . A clear dose-dependent effect was observed when 129 AS was added to each transport medium containing a fixed GITC concentration (Fig. 1D) . Because 130 lower Ct values indicate higher qRT-PCR detection efficiency, these results clearly suggest that 131 adding AS to the transport media can reduce the amount of GITC required for sample denaturation 132 and increase the detection efficiency for SARS-CoV-2 RNA. 133 GITC is commonly used at high concentrations in denaturing viral transport media [17, 21] . 134 However, the sharp increase in demand for large-scale nucleic acid testing is causing a shortage of 135 GITC during the ongoing COVID-19 pandemic. Another issue is that the high GITC concentrations 136 used in the viral testing platforms can react with bleach (sodium hypochlorite) to produce harmful 137 cyanide gas [27] . Therefore, reducing the GITC concentrations in transport media is worthwhile. 138 However, we found that simply reducing its concentration in transport medium decreased the 139 detection efficiency towards SARS-CoV-2 RNA (Fig. 1A and 1B) . Here, we showed that adding AS 140 to the transport media not only recovered but also improved the detection efficiency when low 141 concentrations of GITC were present. The use of AS to precipitate proteins out of solution is known, 142 and AS have been reported to suppress more than 90% of RNase A activity [28, 29] . Therefore, the 143 improved RNA detection efficiency observed in this study possibly resulted from AS-induced 144 denaturation of the RNase A protein, but further research is needed to confirm it. 145 To compare the virus inactivation effects of denaturing transport media containing low 147 concentrations of GITC and 1M AS with a commonly used denaturing transport medium, a plaque 148 reduction assay designed for enveloped and non-enveloped viruses was performed. SARS-CoV-2 is 149 an enveloped virus. However, the biosafety hazards relating to conducting experiments with a live 150 SARS-CoV-2 virus caused us to use a common enveloped virus, HSV-1, to evaluate the inactivation 151 effect of various transport media against this virus. We also tested EV71, a non-enveloped virus with 152 a typical icosahedral capsid structure. After subjecting the viruses to various denaturing solutions at 153 RT for 1 h, each virus was subjected to a plaque reduction assay. 154 The results showed that a common denaturing viral transport medium containing 3M GITC 155 completely inactivated both HSV-1 and EV71 ( Fig. 2A and 2B) . No difference was observed in 156 HSV-1 inactivation when 1M AS was added to the media even when the GITC concentrations were 157 reduced to 0.1M ( Fig. 2A) . In the EV71 inactivation experiments, the effect of supplementing the 158 transport media with 0.5 M GITC plus 1M AS was equal to that where 3M GITC was present, and 159 EV71 was completely inactivated (Fig 2B) . These results suggest that decreasing the GITC 160 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 March 2, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 concentrations but adding AS to the transport media did not affect the virus inactivation effect when 161 compared with general denaturing transport medium containing GITC at high concentration. 162 Faced with the ongoing global COVID-19 pandemic, we sought a safer, low-cost denaturing viral 164 transport medium for preserving SARS-CoV-2 RNA without compromising the effectiveness of viral 165 detection. Based on the components of the modified commercial transport medium we used as a 166 reference reagent, we reduced the GITC concentrations in the media, added AS, and obtained a new 167 formula. We tested the different transport media by detecting SARS-CoV-2 pseudovirus in a throat 168 swab sample and via a viral inactivation assay targeting both enveloped and non-enveloped viruses. 169 We found that adding AS to the denaturing transport media reduced the use of GITC, improved 170 SARS-COV-2 RNA detection, and did not compromise the virus inactivation effect of the media. 171 These findings suggest that AS is a potential component that when added to denaturing transport 172 medium may reduce the cost and improve the detection efficiency towards SARS-COV-2 RNA. 173 Author Xiujing Lu was employed by GBCBIO Technologies Inc.. The remaining authors declare that 175 the research was conducted in the absence of any commercial or financial relationships that could be 176 construed as a potential conflict of interest. 177 LG All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 March 2, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 Reduces GITC for SARS-COV-2 Detection virus. Viruses were incubated in PBS or transport media containing the indicated components, 268 followed by plaque assays on Vero cells. Plaques were visualized by crystal violet staining. Left 269 panels: representative plaques. Right bar charts: virus titres calculated from the plaque assays. Data 270 represent the average ± SD from three independent experiments. 271 272 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 March 2, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 470-473. 192 2. WHO, Weekly epidemiological update on COVID-19 -22 Heavily mutated Omicron variant puts scientists on alert A Comprehensive Review of the Global Efforts on COVID-19 Vaccine Development. ACS Reduces GITC for SARS-COV-2 Detection 6 A Review of the Progress and Challenges of Developing a Vaccine for COVID-19 COVID-19 Antiviral and Treatment Candidates: Current Status Antiviral treatment of COVID-19 Diagnostic performance of different sampling approaches for SARS-CoV-2 RT-206 PCR testing: a systematic review and meta-analysis. 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