key: cord-0297134-2jnm3nx0
authors: Banada, P. P.; Green, R.; Banik, S.; Streck, D.; Montalvan, I.; Reiss, R.; Jones, R.; Marras, S.; Chakravorty, S.; Alland, D.
title: An expanded high throughput RT-PCR assay to rapidly identify all known SARS-CoV-2 variants of concern using melting temperature coding.
date: 2022-01-21
journal: nan
DOI: 10.1101/2022.01.18.22269424
sha: 60cddc8b31f5ffe2fdaa8b1da5bf5b395ed376f8
doc_id: 297134
cord_uid: 2jnm3nx0

Background. The rapid emergence of new vaccine-resistant SARS-CoV-2 variants of concern (VOC) requires an equally rapid deployment of diagnostic tests to specifically identify each VOC as soon as it arises. Here, we report an expanded version of our previously described sloppy molecular beacon (SMB) Alpha/Beta/Gamma RT-PCR melting temperature (Tm) signature-based assay, which now includes modifications that allow specific detection of Delta (B.1.617.2) and Omicron (B.1.529) VOCs. Methods. We developed a dual SMB assay (SMB-452) which targeted the T22917G (L452R) mutation in the SARS-CoV-2 spike protein to specifically detect the Delta VOC. We also identified a Tm profile in our existing SMB-501 and SMB-484 assays (which detect mutations in codons 501 and 484 of the SARS-CoV-2 spike protein, respectively) that differentiate the Omicron-specific N501Y (A23063T) and E484A (A23013C) mutations from both wild type (WT) and other VOCs. The entire six SMB three-codon assay was tested using reference SARS-CoV-2 RNAs. The assay was then validated using clinical samples from COVID-19 patients tested with a LightCycler 480 (LC480) (74 samples), Bio-Rad CFX96 (34 samples), Rotor-Gene Q (Qiagen) (34 samples) and an ABI-7500 (34 samples) RT-PCR instruments. Six SMB Tm results were then inputted into an Excel Analysis tool to generate specific VOC identifications. Results. The limit of detection (LOD) for the new SMB-452 assay, which specifically identified the Delta variant was 1 genomic equivalent (GE) per reaction. The LODs of the SMB-501 and SMB-484 assays which detect Omicron were 100 and 103 GE respectively. Clinical validation of the 3-codon assay in the LC480 instrument showed the assay detected 94% of the samples as WT or VOCs in clinical samples and 6% of the tests producing indeterminate results. None of the samples were incorrectly identified as WT or as a different VOC. Thus, excluding samples with indeterminant results, the assay was 100% sensitive and 100% specific compared to sequencing. There was also 100% concordance between the LC480, BioRad, ABI and Qiagen results excluding negative or indeterminate results; however, the Qiagen assay had significantly more indeterminates than the other assays. Conclusion. This new assay can serve as a robust diagnostic tool for selecting appropriate monoclonal antibody therapy and rapid VOC surveillance.

The global emergence of SARS-CoV-2 variants of concern (VOC), B.1.1.7 (20I, Alpha), B.1.351 (20H, Beta), P.1 (20J, Gamma), B.1.617.2 (21A, Delta), and B.1.1.529 (21K/ Omicron) have been responsible for a series surges in reported COVID-19 cases (1). The Omicron variant, first identified as a VOC by the WHO in November 2021 (2) is now dominating the United States, accounting for 95% of the COVID-19 cases as of Jan 04, 2021 (3) . Several studies have indicated that the Alpha, Beta, Gamma and Delta variants are more transmissible and possibly more virulent than previous SARS-CoV-2 strains (4-7), and the CDC and others have indicated that the Omicron variant may spread twice as rapidly as Delta, but published data are unavailable at the time of this manuscript preparation. The VOCs, and especially Omicron, may confer resistance to therapeutics and decreased vaccine efficacy (7) (8) (9) (10) (11) due to the presence of key mutations in the spike protein (12) (13) (14) (15) (16) . Although the United States has recently increased it capacity to track variants by genome sequencing, only between 2% to 10% of COVID-19 cases are being sequenced (17, 18) and the results of these efforts are often delayed by up to 10 days (19), threatening their utility in preventing further spread or providing real-time therapeutic guidance. A more rapid approach to screen for potential Omicron variants takes advantage of specific RT-PCR COVID-19 tests, which fail to detect Sgene target sequences in Omicron variants. Samples that are negative for the spike target but positive for another SARS-CoV-2 sequence identify presumptive Omicron infections, which are then confirmed by DNA sequencing (1). However, this type of test only identifies Omicron variants, and it still requires sequence confirmation and the associated time delay.

We have previously demonstrated an RT-PCR based method that uses sloppy molecular beacons (SMBs) combined with melting temperature (Tm) code analysis to detect mutations in codons 501 and 484 of the SARS-CoV-2 spike protein. We showed that this assay could specifically identify WT SARS-CoV-2 and the Alpha, Beta and Gamma variants (20) . Delta and Omicron have developed additional variant defining mutations. For Delta, these include the spike protein mutations E156-, F157-, L452R, T478K, D614G, P681R, D950N; for Omicron, these include the spike mutations H69-, V70-, K417N, T478K, . CC-BY-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 January 21, 2022. ; https://doi.org/10.1101/2022.01.18.22269424 doi: medRxiv preprint E484A, N501Y, D614G, P681H. As our earlier assay only targeted mutations in codons 501 and 484 of the spike protein, it was unable to identify Delta variants. However, we expected that a new assay, which could detect mutations at codon 452 should be able to identify Delta variants, and that our existing codon 484 assay would be able to specifically identify Omicron variants if it was able to distinguish the WT 484 sequence present in both WT and Alpha strains from the E484K mutation present in Beta and Gamma, and the E484Q mutation present in Kappa from the E484A mutation present in Omicron (21) . Our codon 501 assay would also be able to provide additional robustness by distinguishing Delta from Omicron since Omicron contains the N501Y mutation also present in Alpha, Beta, and Gamma while Delta does not (5, 12, 22, 23) . Thus, we expected that a simple combination of assays targeting mutations at codons 501, 484, and 452 of the spike protein would be able to specifically identify all the current SARS-CoV-2 VOCs, providing a rapid diagnostic and epidemiological tool.

Here, we describe an expanded assay to identify and distinguish Delta and Omicron variants along with Alpha, Beta, and Gamma, with high specificity and sensitivity. We demonstrate that this approach is flexible and can be used for detecting VOCs using both SARS-CoV-2 RNA and clinical samples. We further confirmed assay performance in four different RT-PCR instruments commonly available in diagnostic laboratories. Adaptation of our assay will enable real-time detection of SARS-CoV-2 variant spread, without the need for whole genome sequencing on all samples.

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The copyright holder for this preprint this version posted January 21, 2022. 

and E484 were used as reported from our earlier publication (20) . An additional assay to detect the L452R (T22917G) mutation present in B.1.617.2 (Delta) was designed similarly as described previously (20) .

Briefly, a total of 412,389 high quality SARS-CoV-2 genome sequences deposited in GISAID (24) as of Feb 19, 2021, were analyzed using BLAST (25) and aligned with MAFFT (26) . Primers and probes were designed on the basis of sequence conservation using Primer3 program (27) Table 1 for an asymmetric one-step RT PCR. A 1 µl of the template RNA was added per 20 µl reaction. 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 January 21, 2022. ; https://doi.org/10.1101/2022.01.18.22269424 doi: medRxiv preprint internal control (IC) to assess failed run and a successful run. An "invalid" call is made if the IC fails to generate a Ct along with the SMB probes and is called "negative" if all probes are negative except for the IC. For VOC classification, the Tm signature values that are generated from each of the six SMB probes . CC-BY-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 January 21, 2022. replicates were positive was found to be 1 GE (Fig. 1A) . The LOD for detecting mutations in codons 501 (SMB-501 assay) and 484 (SMB-484 assay) in our previously published test (20) was re-tested with Omicron RNA in 10-fold dilutions ranging from concentrations of 10 5 -1 GE/reaction ( Fig. 1B and C) in the presence of COVID-19 negative nasal swab matrix. This testing established the SMB-501 assay LOD as 100 GE/reaction (Fig. 1B ) and the SMB-484 assay LOD as 10 3 GE/reaction (Fig. 1C) . Although, 3 out of 4 (75%) at 100 GE/reaction was positive for SMB-484 assay and further work is underway to improve the SMB-484 LOD for Omicron, clinical sensitivity of the assay appears to be excellent (see below).

Tm code definition. The Tm values produced by all SMBs against the reference WT and the MT SARS-CoV-2 strains generated in different instruments are listed in Table 2 . Tm values can vary slightly between samples and different instrument platforms. The 2 or 3-probe Tm coding approach explained previously (20) provides for a robust sequence identification even in the presence of these Tm fluctuations as shown in Table 2 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 January 21, 2022. ; https://doi.org/10.1101/2022.01.18.22269424 doi: medRxiv preprint codes were established for other codons and both WT and variant alleles as also shown in Table 2 . The six-Tm code generated by all three assays was recorded and used as the reference Tm signature for each VOC. Table 2 , a total of 74 confirmed COVID-19 positive patient samples were tested out of which 34 were tested with all three defined assays (SMB-501, SMB-484, and SMB-452) on four different RT-PCR instruments with melt analysis capability. The remaining 40 positive samples and 5 confirmed negative samples were tested with the LC480 only, and these results were used for the primary clinical performance analysis. The sample collection timeline and the initial Ct value at collection is shown in supplementary Fig. 1S . We tested the samples collected over 9 months from April to December 2021, and Ct values ranged from 12 to 37.6. All identification for the VOC were made using the Excel analyze tool. The Tm values obtained from all 3 assays, and identification for each patient sample tested are listed in Table 2 . The clustering of various mutations and the establishment of a Tm signature to identify specific WT or VOCs using SARS-CoV-2 SMB genotyping test, is demonstrated in Fig. 2 . Sanger sequencing of all samples (Table 3 ) was used to confirm the strain identity of each sample. Compared to Sanger Sequencing, the LC480 correctly identified 49/53 (92%) of the Delta variants as Delta, and correctly identified all 13/13 (100%) Omicron variants as Omicron. Of the four samples not identified as Delta by both the assay and Sanger sequencing, three of the specimens were identified as Delta by the SMB-assay, but failed sequencing, and for of the specimens identified as Delta by sequencing produced indeterminate results by the SMB assay ( Table 3) . None of the samples were incorrectly identified as WT or incorrectly identified as a different VOC. Thus, excluding samples with indeterminant results, the assay was 100% sensitive and 100% specific. Including the four indeterminate samples reduced assay sensitivity to 94%, without changing the specificity.

Performance of the other three test instruments were compared to the LC480 as the gold standard.

The BioRadCFX96 identified 97% (33/34) of the samples correctly and one sample tested negative (Kappa, κ=0.9). The ABI7500 also showed a high agreement with the LC480 (κ=0.9), where 32/34 (94%) of the samples were in agreement, and one sample was indeterminate. However, the Qiagen rotor-geneQ (RGQ)

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The copyright holder for this preprint this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.18.22269424 doi: medRxiv preprint instrument produced relatively high indeterminate rates where a variant type could not be called. The RGQ detected 67.6% of the specimen in agreement with LC480 results (κ=0.39) and 11/34 (32.4%) of the samples were indeterminates.

Although whole genome sequencing is a powerful tool to identify new viral lineages, a simple high throughput screening test that accurately identifies VOC provides many advantages. The current study demonstrates that a modification of our simple and easily adoptable assay which was previously developed to identify Alpha, Beta and Gamma strains, can also detect the rapidly emerging Omicron variant. The assay is sensitive, specific and high throughput and can be performed on most qRT-PCR instruments once reference Tm values are established, unlike almost all commercial assays (31, 32) . In our previous publication (20) , we hypothesized that mutations at the codons 501 and 484 would be common in other emergent SARS-CoV-2 variants, and mutations at both codons are repeated in Omicron. Although Delta predominantly remained wild type at these codons, mutations at 452 codon similar to the CAL.20C variant observed first in California (33) , is considered a key Delta-defining mutation. The mutations on these codons have been shown to be responsible for the increased infectivity, transmission, escape humoral immunity and reduced susceptibility to monoclonal antibody treatments (5, 34) and data is not clear yet on their resistance to antivirals. Assays that detect mutations in these codons will help with surveillance to track the variants and may also help guide targeted therapy.

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The copyright holder for this preprint this version posted January 21, 2022. ; Table 1 . Primers, probe, and PCR conditions. The lowercase letter indicates the SMB probe region and the uppercase letter indicate the SMB stem region and pdU -C5 Propynyl-deoxyuridine and BHQ indicates "black hole quencher". . CC-BY-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.

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The copyright holder for this preprint this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.18.22269424 doi: medRxiv preprint Fig. 2 . Variant of concern (VOC) detection using the SARS-COV-2 SMB genotyping test. Correlation plots showing grouping and classification of patient specimens (N=74) tested in Roche LC480 instrument using SMB-501 (blue), SMB-484 (Green) and SMB-452 (Red) assays (A). Identification Tm signature specific for the wild type (WT, B); Alpha (C); Delta (D) and Omicron (E) are highlighted and indicated by arrows.

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