key: cord-0958480-5ek0km9l
authors: Gandotra, N.; Tikhonova, I.; Cheermarla, N. R.; Knight, J.; Foxman, E.; Giraldez, A.; Shen, P.; Bilguvar, K.; Scharfe, C.
title: A two-pronged approach for rapid and high-throughput SARS-CoV-2 nucleic acid testing
date: 2020-12-07
journal: nan
DOI: 10.1101/2020.12.04.20234450
sha: 4115e2fa3a77878a4315fb5bbfdae895f6a66989
doc_id: 958480
cord_uid: 5ek0km9l

Improved molecular screening and diagnostic tools are needed to substantially increase SARS-CoV-2 testing capacity and throughput while reducing the time to receive test results. Here we developed multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) for detection of SARS-CoV-2 using rapid DNA electrophoresis and alternatively using multiplex viral sequencing (mVseq). For RNA specimens extracted from nasopharyngeal (NP) swabs in viral transport media (VTM), our assays achieved a sensitivity for SARS-CoV-2 detection corresponding to cycle threshold (Ct) of 37.2 based on testing of these specimens using quantitative reverse transcription PCR (RT-qPCR). For NP swab-VTM specimens without prior RNA extraction, sensitivity was reduced to Ct of 31.6, which was due to lower concentration of SARS-CoV-2 genome copies in VTM compared to RNA-extracted samples. Assay turnaround time was 60 minutes using rapid gel electrophoresis, 90 minutes using Agilent Bioanalyzer, and 24-48 hours using Illumina sequencing, the latter of which required a second PCR to produce a sequence-ready library using m-RT-PCR products as the template. Our assays can be employed for high-throughput sequencing-based detection of SARS-CoV-2 directly from a clinical specimen without RNA isolation, while ease-of-use and low cost of the electrophoresis-based readout enables screening, particularly in resource-constrained settings.

The emergence of worldwide Coronavirus disease 2019 (COVID-19) has spurred technology development for SARS-CoV-2 molecular diagnostic testing. Traditional viral nucleic acid testing (NAT) has relied on extracting RNA from patient samples such as nasopharyngeal (NP) swabs in viral transport media (VTM) followed by virus detection using RT-qPCR 1, 2 . Scalability of this workflow during this COVID-19 pandemic has been hampered by supply shortages in NP swabs and RT-qPCR assay reagents, and by limited sample throughput capabilities and extended time to report test results. Recently, several innovative approaches for SARS-CoV-2 testing have been developed such as analysis of human saliva specimens with [3] [4] [5] [6] [7] or without a prior RNA purification step 8 , a rapid colorimetric assay 9 or a CRISPR-based assay 10 using reversetranscription loop-mediated isothermal amplification (RT-LAMP), an amplification-free CRISPR-Cas13a-based mobile phone assay 11 , and SARS-CoV-2 detection using next-generation sequencing as a readout [12] [13] [14] [15] . Successful implementation of these approaches into workable testing solutions could dramatically increase SARS-CoV-2 diagnostic capacity and throughput while reducing the time to receive test results.

Here, we developed a two-pronged approach for rapid and for high-throughput SARS-CoV-2 nucleic acid testing (Figure 1 ). Our approach utilizes multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) to capture three SARS-CoV-2 genomic regions plus the human RPP30 control gene in a single-tube reaction (4-plex) directly from a small VTM sample without prior RNA extraction. DNA gel-electrophoresis was developed for the rapid readout of m-RT-PCR amplification products based on their designed size differences. Alternatively, automated DNA fragment detection using the Agilent Bioanalyzer instrument was employed for medium-throughput applications (12-96 samples within 90 minutes). For high-throughput SARS-CoV-2 testing of potentially tens of thousands of samples, m-RT-PCR amplification products were converted into a sequence-ready library 16 for Illumina sequencing with results available within 24-48 hours. Additional improvements of the sequencing workflow for SARS-CoV-2 screening and diagnostic testing will enable increased capacity while reducing the time to receive test results. Major challenges remain at the "front end" of sample acquisition and preprocessing that will need to be addressed for high volume implementation.

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The Yale Institutional Review Board determined this study to be not human subjects research (IRB Protocol #2000029556). Residual nasopharyngeal (NP) swab samples from clinical testing (n=30) were obtained from the Yale-New Haven Hospital (YNHH) Clinical Virology Laboratory.

Samples were de-identified and labeled only with the Ct values from clinical virology testing using RT-qPCR. NP swabs had initially been collected and placed in viral transport media (VTM) by the healthcare provider, and residual VTM was stored at -80C immediately following sample accessioning. Clinical virology testing was previously performed by extracting total nucleic acids using the NUCLISENS easyMAG platform (BioMérieux, France), followed by reverse transcriptase quantitative PCR for N1, N2, and RNAse P targets following the CDC protocol 1 . The 30 samples included 20 positive samples with higher (Ct value for N1, 12.1-24.9) and lower (Ct value for N1, 31.6-37.2) viral loads and 10 negative samples ( Table 1) .

We adopted the protocol described by Ladha et al. 17 . CC-BY-NC 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)

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The four m-RT-PCR amplicons were selected to have different lengths for size separation and analysis using DNA electrophoresis. Volumes of 5 µl or 1 µl of the m-RT-PCR product was run directly on a 1.5% agarose gel or the Agilent Bioanalyzer, respectively (Figure 2) . Alternatively, m-RT-PCR amplicon readout was developed for next-generation sequencing (Figure 3 ).

Producing the sequence-ready library containing the Illumina indexes required a second PCR using the first m-RT-PCR products as the template 16 . This second PCR was run using this protocol: 98 °C for 50 s, 14 cycles of 98 °C for 16 s, 72 °C for 20 s followed by a final extension of 72 °C for 2 min. A 10 µl aliquot of the 2 nd PCR amplification products from each sample (n=30) was pooled and bead-purified using a 1.8:1 ratio of Ampure XP beads (Beckman Coulter) to DNA. Following quantification, a small aliquot of the library (18 µl of 1.3 nM final concentration) was run on Illumina NovaSeq 6000 or MiSeq using paired-end 150 bp sequencing. The paired-end reads were aligned using BWA MEM to a reference containing the SARS-CoV-2 genome (NC_045512.2) and RPP30 gene (NM_001104546.1). An in-house script parsed the alignments, and, for each pair, computed the alignments' starting locations and strands for the pair's two reads. Read pairs with unique alignments within 4bp of the expected amplicon endpoints, with proper alignment directionality, were counted as read pairs for that amplicon. Other reads were filtered from the counts.

A single-tube multiplex RT-PCR (4-plex) was developed to capture three regions of the SARS-CoV-2 genome and the Human RPP30 control amplicon from NP swab-VTM specimens. The assay was first evaluated for NP swab-VTM specimens without prior RNA extraction. Two alternative approaches for readout of m-RT-PCR amplicons were developed (Figure 1 ).

First, a fast readout was designed to return results in less than 60 minutes by analyzing the m-RT-PCR amplification products directly using agarose gel electrophoresis. SARS-CoV-2 positive VTM samples showed three bands corresponding to the three viral amplicons (Figure 2A) , while negative samples only showed amplification of the single RPP30 target ( Figure 2B) . The reagent and supplies cost of the gel-based assay including QE buffer, PCR reagents, primers, tubes, pipet tips, gloves, and gel was estimated at $5.37 per sample. Alternatively, m-RT-PCR . CC-BY-NC 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 December 7, 2020. ; https://doi.org/10.1101/2020.12.04.20234450 doi: medRxiv preprint amplification products could also be analyzed using the Agilent Bioanalyzer within 90 minutes, and with an ability to multiplex larger samples batches (e.g., 96 samples using the Agilent TapeStation, $1.69 per sample) at an estimated reagent and supplies cost of $7.01 per sample.

Using the Bioanalyzer, SARS-CoV-2 positive samples showed four peaks including the human RPP30 amplicon at a size of 145 bp, and the three viral amplicons of 157, 210 and 348 bp ( Figure 2C) . Negative samples showed only a single peak for RPP30 ( Figure 2D) . For some samples, additional smaller peaks were seen indicating non-specific amplification products or primer dimers, which however differed in size compared to the intended targets and could thus be readily identified. We found that all NP swab-VTM specimens with higher viral loads (Ct N1, 12.1-24.9) consistently showed the three SARS-CoV-2 bands on the agarose gel and four peaks on the Bioanalyzer. Among the samples with lower viral loads (Ct N1, 31.6-37.2), only one sample (S15, Ct N1, 31.6) showed a single SARS-CoV-2 and the RPP30 peak on the Bioanalyzer (Supplemental Figure 1) but not on the agarose gel. The other nine low viral-load samples did not show visible amplification products using either detection method.

Second, high-throughput readout of m-RT-PCR products using next-generation sequencing was developed ( Figure 3A ). Similar to results above, we found that VTM samples with high viral loads consistently generated a sufficient number of reads for at least one SARS-CoV-2 amplicon and for RPP30, while samples with lower viral load did not show read counts that were significantly different from the small level of background reads in negative control samples. All 30 samples were processed together with an estimated reagent cost of $6.85 per sample and a turnaround time of 24-48 hours from VTM sample-in to sequencing data results-out. Turnaround for generating sequencing data could be reduced to less than 24 hours by using very short read sequencing 12 , which however would require additional evaluation of our bioinformatics pipeline.

We were then interested to evaluate the performance of mVseq for RNA extracted from NP swab-VTM specimens. Extracted RNA has been the standard input specimen for RT-qPCR assays in the clinical laboratory 2 . We extracted RNA from NP swab-VTM specimens for eight samples with lower viral load (Ct N1, 31.6-37.2) and from one sample with lower Ct N1 of 20.1 ( Table 1) 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 December 7, 2020. ; https://doi.org/10.1101/2020.12.04.20234450 doi: medRxiv preprint peak and the human RPP30 peak (Supplemental Figure 2) . Using sequencing-based detection, four of the eight low-viral load samples (S11, S13, S14, S15) were found positive for all four m-RT-PCR amplicons, three samples (S12, S17, S18) were positive for two viral amplicons and RPP30, while one sample (S16) was positive for only one viral amplicon and RPP30 ( Figure 3B) . Thus, using extracted RNA from NP swab-VTM samples as the input specimen, our mVseq assay was able to correctly detect all positive samples that had an N1 Ct value of 37.2 or lower by the clinical RT-qPCR assay. The reduced sensitivity for using NP swab-VTM specimens directly without the RNA extraction step was likely due to the smaller amount of viral copies added into the m-RT-PCR. The maximum volume of NP swab-VTM specimen that could be added to the m-RT-PCR was 5.5 µl (e.g., 10 .99 µl of VTM/QE mixture).

For comparison in the clinical laboratory protocol, the 5 µl of extracted RNA used in testing correspond to 20 µl of NP swab-VTM specimens (a ~4-fold increased concentration) as 200 µl of the primary NP swab-VTM specimens are eluted into 50 µl during RNA purification. In future experiments, VTM/QE mixture samples could be concentrated (f.e., using magnetic beads) to increase assay sensitivity for detecting lower viral-loads from a primary clinical specimen.

A major limitation of the m-RT-PCR-based approach is the need to open tubes for SARS-CoV-2 amplicon detection and sequencing. In comparison, viral nucleic acid testing using goldstandard RT-qPCR does not require opening tubes after amplification and thus greatly minimizes the risk for cross contamination. Additionally, many COVID-19 samples have very high viral loads (Ct value for N1 gene of 7-15) and cross contamination between samples is a major concern. To minimize contamination, we followed procedures established for working in clinical molecular laboratories including separation of pre-PCR and post-PCR laboratory space, the use of a dedicated hood for making dilutions, sterilizing Eppendorf pipettes with 10% bleach followed by UV-light treatment for 30 minutes. While a small number of background reads was seen in negative controls (<10 reads, Figure 3A ), those could be distinguished from the multifold higher amplicon read levels in test samples. To avoid cross contamination during consecutive sequencing runs, instruments were washed with bleach according to Illumina's guidelines before every run 12, 15 .

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Improved molecular screening and diagnostic tools are needed to substantially increase SARS-CoV-2 testing capacity and throughput while reducing the time to receive test results. Here we developed a two-pronged approach for SARS-CoV-2 nucleic acid testing for two common detection technologies. This choice was made to provide new solutions for both rapid (using DNA electrophoresis) and high-throughput (using sequencing) readouts for SARS-CoV-2 nucleic acid testing. Notably, both readout modalities are coupled with the same multiplex RT-PCR assay, which is flexible and scalable to incorporate the larger SARS-CoV-2 genome sequence and additional RNA viruses. For RNA extraction followed by multiplex RT-PCR, all positive samples with a N1 Ct value of 37.2 or lower could be detected using either sequencing or the Bioanalyzer as readout. Eliminating the initial RNA extraction reduced the sensitivity for both modalities, and we were still able to accurately detect SARS-CoV-2 in all specimens with high viral loads. We envision the low cost and ease-of-use of the DNA electrophoresis-based readout as an attractive solution for rapid screening, and particularly in settings with constrained resources.

We thank Annie Wylie and Nathan Grubaugh for providing synthetic viral RNA fragments, and Shrikant Mane and the YCGA staff for their support and advice with this project.

NG, CS designed the assay, NG performed the experiments and developed the assay, EF and NC provided clinical samples, IT extracted and provided RNA specimens, JK established the pipeline for sequence data analysis, KB, AG, and PS provided assay reagents and advise, and NG and CS wrote the manuscript, which all authors edited and approved.

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The copyright holder for this preprint this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.04.20234450 doi: medRxiv preprint . CC-BY-NC 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 December 7, 2020. ; https://doi.org/10.1101/2020.12.04.20234450 doi: medRxiv preprint . CC-BY-NC 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.

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The copyright holder for this preprint this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.04.20234450 doi: medRxiv preprint Supplemental Figure 1 : Detection of a low SARS-CoV-2 viral load sample from VTM. Agilent Bioanalyzer profile of a NP swab-VTM specimen with low viral loads (S15, Ct 31.6) shows amplification peaks for SARS-CoV-2 (Cov-310) and the human RPP30 control gene.

Agilent Bioanalyzer profiles of RNA specimens extracted from NP swab-VTM specimens with low viral loads (31.6-37.2) show at least one amplification peak for SARS-CoV-2 and RPP30 control gene. In comparison, the profile of the RNA specimen from a high viral load sample in panel 9 (S01, Ct 20.1) shows four peaks including the human RPP30 amplicon and the three SARS-CoV-2 amplicons.

Cov-310

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