key: cord-0964810-9x0jyah2
authors: Sahajpal, N. S.; Mondal, A. K.; Njau, A.; Ananth, S.; Kothandaraman, A.; Hegde, M.; Chaubey, A.; Padala, S.; Kota, V.; Caspary, K.; Tompkins, S. M.; Ross, T. M.; Rojiani, A. M.; Kolhe, R.
title: Clinical validation of innovative, low cost, kit-free, RNA processing protocol for RT-PCR based COVID-19 testing.
date: 2020-07-30
journal: nan
DOI: 10.1101/2020.07.28.20163626
sha: 70ca5d8b58bca2cd2cf0615e470513819c40577c
doc_id: 964810
cord_uid: 9x0jyah2

The current gold-standard molecular diagnosis for COVID-19 is based on a multi-step assay involving RNA-extraction and RT-PCR analysis for the detection of SARS-CoV-2. RNA-extraction step has been a major rate-limiting step in implementing high-throughput screening for COVID-19 during this pandemic. Moreover, clinical laboratories are facing several challenges that include cost, reagents, instrumentation, turn-around time, trained personnel, and supply-chain constraints to efficiently implement and sustain testing. Cognizant of these limitations, we evaluated the extraction-free methods described in the literature and have developed an innovative, simplified and easy protocol employing limited reagents to extract RNA for subsequent RT-PCR analysis. Nasopharyngeal-swab samples were subjected to the following individual conditions: 65{degrees}C for 15 minutes; 80{degrees}C for 5 minutes; 90{degrees}C for 5 minutes or 80{degrees}C for 1 minute, and processed for direct RT-PCR. These groups were also compared with a supplemental protocol adding isopropanol-ethanol-water elution steps followed by RT-PCR assay. The direct RT-PCR assay did not detect SARS-CoV-2 within the various temperature incubation only groups, whereas, the 90{degrees}C for 5 minutes-isopropanol-ethanol-water method was found to be comparable to the FDA-EUA method. Evaluation of the performance metrics for 100 clinical samples demonstrated a sensitivity of 94.2% and a specificity of 100%. The limit of detection was ascertained to be ~40 copies/ml by absolute-quantification. The protocol presented for this assay employs limited reagents and yields results with high sensitivity. Additionally, it presents a simplified methodology that would be easier to implement in laboratories in limited resource countries in order to meet the high current COVID-19 testing needs.

detection was ascertained to be ~40 copies/ml by absolute-quantification. The protocol presented 48 for this assay employs limited reagents and yields results with high sensitivity. Additionally, it 49 presents a simplified methodology that would be easier to implement in laboratories in limited 50 resource countries in order to meet the high current COVID-19 testing needs. 51 52 . 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. (1) . Among these strategies, the 62 most critical method adopted to measure and contain its spread is testing for SARS-CoV-2, 63 typically utilizing nasopharyngeal swab specimens (2). Diagnostic testing for SARS-CoV-2 has 64 been under intense scrutiny due to its tremendous, immediate clinical and epidemiologic 65 significance in the current COVID-19 pandemic. The global demand for testing has reached a 66 crisis level , with clearly identifiable regional disparities. At present, RT RT-PCR.pdf, last accessed July 12, 2020).

The RT-PCR based methods employ sophisticated commercial kit based RNA extraction 72 protocols followed by a one-step RT-PCR assay for the detection of the SARS-CoV-2 virus.

However, there are several challenges in the implementation and sustainability of this method for 74 COVID-19 testing, especially in resource-limited countries. The technical and financial 75 challenges include the cost of reagents/ kits, equipment, turnaround time, and trained personnel.

The RNA extraction step is a bottleneck by virtue of its requirement of expensive kits, a 77 processing time of ~100 minutes with automated instrumentation, and trained personnel to run 78 the protocol. For laboratories that employ manual methods of RNA extraction, this process could 79 take much longer, up to several hours. Further, COVID-19 testing has lagged significantly even 80 in developed countries, owing to the widespread supply chain constraints of these kits.

In these circumstances, there is a dire need to identify solutions or alternatives to these 82 limitations and identify methods that can be easily implemented, with simple protocols,

. 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 30, 2020. CoV-2 (3-6). Both protocols typically employ heat-shock treatment of the samples before 89 proceeding to the RT-PCR step. However, there are several limitations to both these concepts.

The dry-swabs collected in TE require immediate processing, which does not seem to be a viable Assay for the detection of SARS-CoV-2 (FDA-EUA Method) 105 The assay is based on RNA extraction followed by TaqMan-based RT-PCR assay to conduct the 

. 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 30, 2020. . https://doi.org/10.1101/2020.07.28.20163626 doi: medRxiv preprint 116 The RNA extraction is semi-automated and occurs in a 96-well plate format. In brief, an aliquot 117 of 300µl from each sample, including positive and negative controls, were added to respective In preliminary experiments, we evaluated two concepts: a. Heat-shock treatment of NPS 128 samples for direct RT-PCR assay; b. Heat-shock or lysis buffer treatment of NPS samples 129 followed by RNA extraction using limited reagents, followed by RT-PCR assay. An aliquot of 130 NPS sample was subjected to heat-shock treatment using the Hybex Microsample Incubator 131 (Scigene). The NPS samples were subjected to the following conditions: 65° C for 15 minutes; 132 80° C for 5 minutes; 90° C for 5 minutes; 80° C for 1 minute, and 40 µl of the sample was 133 directly processed for RT-PCR assay(7-8). Additionally, the same sample was assessed for RNA 134 extraction using an alternate method. In this method, after the respective heat-shock treatment, 135 300 µl of the sample was processed for nucleic acid precipitation using 300 µl isopropanol, 136 followed by 75% ethanol wash, and finally dissolving the nucleic acid in 50 µl water 137 (isopropanol-ethanol-water). Similarly, 40 µl was used for the RT-PCR assay. In addition to the 138 heat-shock method, we also treated the NPS samples with RBC lysis buffer with (56° C for 5 139 minutes) or without heat-shock treatment, followed by isopropanol-ethanol-water steps.

Clinical sample evaluation using an alternate method 141 From the above-described pilot studies, the method with the highest sensitivity was chosen for 142 analytical evaluation (sensitivity, specificity, accuracy, and precision) using previously 143 confirmed positive (n=50) and negative (n=20) samples. Also, the limit of detection was 144 determined by absolute quantification RT-PCR analysis.

. 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 30, 2020. Isopropanol-Ethanol-Water > 90° C for 5 minutes-Isopropanol-Ethanol-Water (Fig 1) . The 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 30, 2020. . https://doi.org/10.1101/2020.07.28.20163626 doi: medRxiv preprint Fifty previously confirmed positive and twenty negative samples were processed for RNA 176 extraction by the alternate method and RT-PCR analysis. Analytical sensitivity of 92%, 177 analytical specificity of 100%, accuracy of 94.2%, and precision of 100% were observed (Table   178 1). The Ct value difference for both N and ORF1ab genes is depicted in Fig 2 and 3 . The LOD 179 was determined to be ~40 copies/ml by absolute quantification calculation. In our pilot studies, we first evaluated the extraction-free RT-PCR methods, where the NSP 204 samples were subjected to different heat-shock treatment (65° C for 15 minutes; 80° C for 5 205 minutes; 90° C for 5 minutes; 80° C for 1 minute), and processed for direct RT-PCR assay. 7,8 206 . 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 30, 2020. . https://doi.org/10.1101/2020.07.28.20163626 doi: medRxiv preprint Although, we used samples with low Ct values (via standard initial testing), no amplification/ 207 detection was observed at all four temperature conditions. This led us to process samples with an 208 alternate protocol, where after heat-shock treatment, the nucleic acid was precipitated with 209 isopropanol, washed with ethanol, and dissolved in water. Additionally lysis buffer was also 210 assessed as an alternative to heat-shock treatment, followed by isopropanol-ethanol-water steps.

Comparing the different heat-shock treatments vs lysis conditions followed by isopropanol-212 ethanol-water steps, the 90° C for 5 minutes followed by Isopropanol-Ethanol-Water method was The implementation of this proposed alternate protocol for COVID-19 has the potential to 231 increase SARS-CoV-2 testing, reducing turnaround times, clearing backlogged samples, and 232 ensuring enormous savings on RNA extraction and/or testing kits and laboratory supplies that are 233 in short supply. This would relieve the pressure mounting on laboratories for increased testing, 234 hopefully making a significant contribution to control of this pandemic.

. 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 30, 2020. . https://doi.org/10.1101/2020.07.28.20163626 doi: medRxiv preprint . 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 30, 2020. . https://doi.org/10.1101/2020.07.28.20163626 doi: medRxiv preprint . 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 30, 2020. 

COVID-19: towards controlling of 238 a pandemic

Report from the American Society for Microbiology COVID-19 international 241 summit, 23 march 2020: value of diagnostic testing for SARS-CoV-2/COVID-19

Improved and Simplified Diagnosis of Covid-19 using TE Extraction from Dry 245

Preliminary support for a "dry swab, 248 extraction free" protocol for SARS-CoV-2 testing via RT-qPCR

Massive and rapid COVID-19 testing is feasible 252 by extraction-free SARS-CoV-2 RT-qPCR

Rapid direct nucleic acid amplification test 255 without RNA extraction for SARS-CoV-2 using a portable PCR thermocycler

Inactivation of coronaviruses by heat