key: cord-0776936-bdfq9m4v authors: Yaniv, K.; Ozer, E.; Plotkin, N.; Bhandarkar, N. S.; Kushmaro, A. title: RT-qPCR assay for detection of British (B.1.1.7) and South Africa (B.1.351) variants of SARS-CoV-2 date: 2021-03-01 journal: nan DOI: 10.1101/2021.02.25.21252454 sha: 9144ab2abfd45ee69f2263c554cae489b09cc4cb doc_id: 776936 cord_uid: bdfq9m4v Less than a year following the SARS-CoV-2 outbreak, variants of concern have emerged in the form of the British variant B.1.1.7 and the South Africa variant B.1.351. Due to their high infectivity and morbidity, it is crucial to quickly and effectively detect them. Current methods of detection are either time-consuming, expensive or indirect. Here, we report the development of a rapid, cost-effective and direct RT-qPCR method for detection of the two variants of concern. We developed and validate a detection system for the detection of the B.1.1.7 variant and another single detection set for the B.1.351 variant. The developed approach was characterized and tested on wastewater samples and illustrated that all primers and probes were sensitive and specific. The novel system presented here will allow proper response and pandemic containment with regard to these variants. In addition, it may provide a basis for developing tools for the detection of additional variants of concern. The SARS-CoV-2 world pandemic erupted early 2020 with rising numbers in morbidity and mortality. SARS-CoV-2 was recognized as an RNA virus, therefore detection methods emerging for immediate response were mainly in the form of reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) . In RT-qPCR, RNA is extracted, undergoes reverse transcription for DNA strand generation, followed by PCR amplification and TaqMan probes fluorescence detection. To date, RT-qPCR is the most common methodology for SARS-CoV-2 diagnostics (Vogels et al., 2020) . Starting in September 2020, new variants of concern of SARS-CoV-2 virus began to emerge. Amongst them, the variants termed the British variant B.1.1.7 and South Africa (SA) variant B.1.351 became dominant compared to the original SARS-CoV-2 virus . Due to their higher infection rate and high morbidity, identification of these variants became essential. A diagnostic tool that could quickly and efficiently distinguish between the variants is imperative to help evaluate the variants' distribution. Proper "variant mapping" will provide much needed information to enforce appropriate policy for pandemic containment. Currently, three methodologies have been developed for SARS-CoV-2 variant diagnostics. The first methodology, that is still mainly being employed, is the next generation sequencing (NGS) approach (Andrés et al., 2020; Khan et al., 2020) . In NGS the entire variant's genome is sequenced. Despite demonstrating the importance of this technique for identification of new variants, its use on previously sequenced variants is time-consuming and requires significant financial means. Additional detection methods are based on RT-qPCR and include a "drop-out" signal, available in commercial kits (such as TaqPath COVID-19 diagnostic tests, Thermo Scientific, Helix ® COVID-19 Test) or a method published in a recent study (Vogels et al., 2021) . These use RT-qPCR with two different markers, a double signal manifest for the original SARS-CoV-2 virus, while only a single signal manifest for the targeted variant. Another detection methodology is through characterization of ΔCt between one detection signal and another amongst the different variants (Kovacova et al., 2021) . Thus theses current RT-qPCR approaches for variant detection, though significantly faster and cost-effective than the NGS methodology, focuses on indirect detection and may result in false/inconclusive identification. 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 1, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 Therefor there is still a great need for a quick improved specificity-and sensitivity SARS-CoV-2 variants detection methods. Such advanced methodologies will be amenable for clinical diagnostics, as well as for environmental-derived quantification, greatly improving wastewater and population level epidemiology. In this study, we developed a RT-qPCR assay for the direct detection of SARS-CoV-2 British variant B.1.1.7 and another set of RT-qPCR primersprobe for the detection of SARS-CoV-2 SA variant B.1.351. Our design was tested on S gene deletion and non-deletion DNA templates and RNA originating from wastewater samples to assess the sensitivity and specificity of the described sets. The original sequence of SARS-CoV-2 (NC_045512.2) was taken from NCBI database. British B.1.1.7 variant (EPI_ISL_742238) and SA B.1.351 variant (EPI_ISL_736935) sequences were taken from GISAID database (Shu and McCauley, 2017) . The probe design focused on the S gene 21724-21828 bp location that includes the British deletion 69-70 or S gene 22243-22331 bp location that includes the SA deletion 241-243. All primers and probes were purchased through Integrated DNA Technologies (IDT). ZEN Quencher was added to the probes as a second, internal quencher in qPCR 5'-nuclease assay. To allow a possibility for duplex assay, S1 probe was assigned a 6-carboxy-fluorescein (FAM) fluorophore and S∆69 probe was assigned to Yakima Yellow (YakYel) fluorophore. S∆241 probe was assigned with FAM as well. RT-qPCR was executed using One Step PrimeScript III RT-qPCR mix using standard manufacture protocol (RR600 TAKARA, Japan). Each reaction mixture contains primers (0.5 µM each), probe (0.2 µM each), ROX reference dye and 5 μL of DNA or RNA (dH2O was added to a final volume of 20 µL reaction volume). RT-qPCR amplification was executed using Step One Plus real-time PCR system (Applied Biosystems, Thermo Scientific). In addition to what is described above, in each run all RT-qPCR experiments included quality controls. The first control was using water sample instead of DNA/RNA (Non template control (NTC)). The second control, used for RNA extractions, was MS2 phage detection (Dreier et al., 2005) . 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 1, 2021. ; https://doi.org/10.1101/2021.02.25.21252454 doi: medRxiv preprint Calibration curves were performed on a known-positive DNA gene block. Two different gene blocks were used; one containing SARS-CoV-2 S gene sequence as reported for Wuhan-Hu-1 (NC_045512.2), the second containing S gene sequence matching the reported 69-70 deletion of the British variant (B.1.1.7) as well as the reported 241-243 deletion of the SA variant (B.1.351). Calibration of S1 probe was performed using S gene sequence from NC_045512.2, while calibration of S∆69 probe and S∆241 was performed using S gene sequence with the relevant deletions. Serial dilutions for the relevant gene block were prepared based on copy number calculations. The resulted Ct values were plotted against the log copy number of the S gene template. Linear regression was performed between the log copy number and the Ct values from the RT-qPCR results. For wastewater sampling, composite sewage samples from the wastewater treatment plant (WWTP) were immediately transferred to the lab under chilled conditions. The samples were kept at 4 o C until processed. Direct RNA was extracted according to manufacture protocol as it describes in NucleoSpin RNA extraction kit (Macherey Nagel, Germany). An amount of 10 5 copies of the phage was added to the lysis buffer in each RNA extraction for inner control. RNA was eluted with 50 μL of RNase free water and kept at -80 o C. Extracted RNA from wastewater sample, known to be SARS-CoV-2 negative, was supplemented with known concentrations of a desired gene block. The samples underwent the same RT-qPCR conditions as described for the calibration curves. Results were plotted to represent the new probes limit of detection in a complex environment. Developing our assay, we focused on the most dominant variants of SARS-CoV-2 currently known. These, the British variant B.1.1.7 and South Africa (SA) variant B.1.351, were deemed the most urgent variants in need for fast detection. Our design for RT-qPCR detection assays of the two variants (Figure 1) , is based on the differences in the S gene from the original 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. completely identical, apart from 6 nucleotides deletions (Fig. 1a) . Our main attempt was to create two separate detections to the amplified area, one corresponding to the original sequence (when using S1 probe) and the other corresponding to the B.1.1.7 (when using the S∆69 probe). Using designated primers (Fig. 1b) to amplify the specified region surrounding the 6 nucleotides differences, an amplification will be generated regardless to the variant. The probes can thus be used in a single duplex assay via separate wavelengths, where a signal signifies a direct detection of either the original sequence or of B.1.1.7. For B.1.351 detection, the designated detection region was chosen further along the S gene when compared to the B.1.1.7 detection region. Focusing on S gene 22243-22331 bp of the original sequence, the original SARS-CoV-2 sequence is identical to the B.1.351 sequence apart from a 9 nucleotides deletion (Fig. 1a) . Using a detection set comprised of two primers meant to amplify the target region, a single probe (S∆241 probe) was designed for the detection of the B.1.351 variant. The S∆241 probe is meant to correspond only to the deletion of 9 nucleotides in 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 1, 2021. ; https://doi.org/10.1101/2021.02.25.21252454 doi: medRxiv preprint the specified region, characterizing B.1.315, therefore will signal detection only when B.1.351 is present and will not correspond to the original sequence. To ensure functionality, the described sets of primers and probes underwent characterization. Initially, a calibration curve was generated for primers with the relevant probe separately, using dsDNA as a template. A detection range of between 10 7 copies and ten copies per µL was tested for each probe. Results plotted for both probes confirmed the chosen primers' (4 different primers, 2 different amplification sets) ability to amplify the target region (Fig. 2) . Furthermore, linear regression performed for both probes demonstrated strong coloration and the probes validity for usage on the amplified fragment. A limit of detection (LOD) could be determined for each probe and was identified as 10 1 copies per µL for all three probes. Ct Log copy number S∆69 S∆241 S1 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 1, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 wastewater detection is important with regards to the development of a quick early warning system for virus detection during the global pandemic (Bar-Or et al., 2020) , wastewater matrix collected from wastewater treatment plant at the city of Beer Sheva, Israel, was chosen as complex environment. All three probes were employed on wastewater samples pre-determined as negative for SARS-CoV-2 with various dsDNA template copies (Fig. 3) . As can be seen in Figure 3 , despite the wastewater matrix, the 3 designed probes displayed high detection sensitivity. With the ability to detect up to 10 1 copies per µL, the new probes demonstrated satisfying detection ability when compared to previously described primers and probes sets for clinical diagnostics (Vogels et al., 2020) . : Lower detection limit of S∆69, S∆241 and S1 primer-probe sets in wastewater matrix. RNA extracted from negative detection wastewater sample (No virus) spiked with known concentrations of SARS-CoV-2 S gene template (10 1 -10 3 S gene template copies μl -1 ) and Non-Template Control (NTC, water). For S∆69 probe and S∆241 probe, the S gene deletion template corresponded to ∆69-70 deletion site in British B.1.1.7 and ∆241-243 deletion site in South Africa B.1.135. For S1 probe, the S gene template corresponded to NC_045512.2 sequence. ND -not detected. Solid lines indicate the median and dashed lines indicate the detection limit as decided by clinical guidelines. 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 1, 2021. ; https://doi.org/10. 1101 To examine the probes specificity and rule out possible false-positive cases, each probe was tested with a negative control. For S1 probe, the negative control was comprised of a dsDNA template with the S gene with ∆69-70 and ∆241-243 nucleotides deletions. While for S∆69 probe and S∆241 probe, the original S gene sequence was used as negative control. As expected, none of the probes manifested a signal in the presence of a negative control and nonspecific detection was not observed (Table 1) . Finally, the three probes, S, S∆69 and S∆241 were tested on wastewater samples (Table 1 ). Moreover, the CDC's N2 detection set was used as standard detection reference that can correspond to each of the variants . According to the results, samples collected at Looking at the detection results from wastewater from Beer-Sheva, Israel, an interesting observation was seeing that the N gene detection constantly produced lower Ct values compared to the S gene detection (with either S1 probe or S∆69 probe). This may imply different gene expression distributions or different durability of the RNA segments, however this needs further study and validation to better understand such an observation. In the meantime, this phenomenon may also affect the "drop-out" assays resulting in false-positives, reinforcing the need in direct detection. Overall, the displayed results indicate that the developed assay can be employed and provide essential, direct detection abilities for the two variants. 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 ongoing concern regarding the COVID-19 pandemic and the emergence of new variants with higher infection rate and morbidity, create great global concern. With regards to the highly dominant variants of concern, British variant B.1.1.7 and South Africa variant B.1.351, current diagnostic tools are expensive, time-consuming or indirect. Here we present an RT-qPCR assay developed for the direct detection of these two variants. An RT-qPCR assay was developed for the direct detection of the British variant (B.1.1.7) and its differentiation from the original SARS-CoV-2 (NC_045512.2). Using a single set comprised of two new primers, two new probes were designed and validated, focusing on the characterized deletion area known as ∆69-70. In addition, an RT-qPCR direct detection assay was developed for the South Africa variant (B.1.351), using two new primers focusing on a characterized deletion area known as ∆241-243, and a third probe was designed and validated. The presented primers and probe sets may be used as described here, or even combined in the future in different combinatorial approaches for rapid, cost-effective and direct detection of the two variants. 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 1, 2021. ; https://doi.org/10. 1101 Naturally occurring SARS-CoV-2 gene deletions close to the spike S1/S2 cleavage site in the viral quasispecies of COVID19 patients Regressing SARS-CoV-2 sewage measurements onto COVID-19 burden in the population: A proof-of-concept for quantitative environmental surveillance Use of bacteriophage MS2 as an internal control in viral reverse transcription-PCR assays Comparative genome analysis of novel coronavirus (SARS-CoV-2) from different geographical locations and the effect of mutations on major target proteins: An in silico insight 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 A novel, room temperature-stable, multiplexed RT-qPCR assay to distinguish lineage B.1.1.7 from the remaining SARS-CoV-2 lineages US CDC real-time reverse transcription PCR panel for detection of severe acute respiratory syndrome Coronavirus 2 GISAID: Global initiative on sharing all influenza data -from vision to reality Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT-qPCR primerprobe sets PCR assay to enhance global surveillance for SARS-CoV-2 variants of concern We would like to acknowledge funding from Ben Gurion University, The Corona Challenge Covid-19 (https://in.bgu.ac.il/en/corona-challenge/Pages/default.aspx) and funding from the Israeli ministry of Health. 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 1, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021