key: cord-0253650-6l07833c authors: Yaniv, K.; Ozer, E.; Kushmaro, A. title: SARS-CoV-2 variants of concern, Gamma (P.1) and Delta (B.1.617), sensitive detection and quantification in wastewater employing direct RT-qPCR date: 2021-07-16 journal: nan DOI: 10.1101/2021.07.14.21260495 sha: b791ccd10eba7cbfbd0bc2ab1304f05c7b9510bf doc_id: 253650 cord_uid: 6l07833c SARS-CoV-2 variants of concern present a worldwide threat. Demonstrating higher infection rate and durability to antibodies when compared to the original SARS-CoV-2 virus, the variants of concern are responsible for continuing global outbreaks. Prompt identification of the infecting SARS-CoV-2 variant is essential for pandemic assessment and containment. However, variant identification is mainly being performed using expensive, time-consuming next generation sequencing. Rapid identification methodology for variants of concern is of great need and various variant-specific assays are being developed. Amongst the variants of concern that have recently appeared, the Gamma variant (P.1, Brazilian) and Delta variant (B.1.617, Indian) are the most prominent. Here we describe the development of a sensitive RT-qPCR assay for the quick direct detection of the Gamma and Delta variants as part of a methodical characterization and detection in municipal wastewater. Since declaring COVID-19 a world pandemic in March 2020, the disease continues to evolve and affect significant portion of the world population. Increasing circulation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus within the world population, results in a continued natural processes of random mutations and evolution 1 . In some cases, a mutation will have an evolutionary advantage and create a new viral lineage that will overpower previous forms. First evidence for such an evolutionary event for the SARS-CoV-2 virus was in April 2020, when the D614G substitution enhanced infectivity and transmission. The D614G mutation lineage became the dominant SARS-CoV-2 variant form 2,3 . By late 2020, the term 'variants of concern' emerged with regards to the high-numbered mutations in a single variant and the increased pathogenicity 4 . Variants of concern are mainly characterized through mutations in their spike protein gene (S gene). Such mutations affect the receptor biding domain affinity 5 and antibody neutralization efficiency 6 . Gamma (P.1, Brazilian) and Delta (B.1.617, Indian) variants of concern were first detected in January 2021 and December 2020 respectively, and by now circulate globally 7 . High transmission rates and lower vaccine efficacy towards these variants 8-10 present a higher threat compared to the original virus. Therefore, quick detection of recognized variants of concern is vital for initiating response and proper policy for pandemic containment. Currently, SARS-CoV-2 variant detection mainly relies on costly next generation sequencing, where results are obtained within 3-5 days 11, 12 . With new variants of interest constantly emerging, sequencing-based approaches are important for their identification, however they present difficulties considering special instruments and resources, including data analysis skills required. To ease on detection a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR)-based assay can provide rapid results for detection of known variants of concern detection. Such an assay can help assess a variant's frequency within the population and contribute to policy guidelines. Indeed a number of publications targeting variants of concern detection using RT-qPCR [13] [14] [15] [16] [17] [18] as well as available commercial kits (TaqPath ThermoScientific, GT molecular, PerkinElmer, etc.), have emerged. Urban morbidity monitoring through wastewater is a known practice 19 . Without the need for cooperation by individuals, it is possible to receive viable important information regarding the spread of a variant in population in a given area. Since erupting, the SARS-CoV-2 virus has been monitored in wastewater around the world [20] [21] [22] [23] [24] [25] . In order to detect specific variants such as the Gamma and Delta variants of concern though, a direct and sensitive probe-based RT-qPCR assay with detection ability in wastewater 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 July 16, 2021. ; https://doi.org/10.1101/2021.07.14.21260495 doi: medRxiv preprint samples is of great need and could help generate better health policies once employed in a given region. In this study, we present a sensitive RT-qPCR assay design for the direct detection of Gamma and Delta variants of concern and its employment in wastewater sampling. The original sequence of SARS-CoV-2 (NC_045512.2) was taken from NCBI database. Gamma RT-qPCR was executed as previously described 17 . Reaction final volume is 20 µL with primers concentration of 0.5 µM and probe concentration of 0.2 µM. The reaction contained 5 µL of RNA sample and ROX as a reference dye. Reaction steps was executed according to manufacture recommended protocol (One Step PrimeScript III RT-qPCR mix RR600 TAKARA, Japan) using Applied Biosystems, Thermo Scientific). Each RT-qPCR run included relevant quality controls, Non template control (NTC) and MS2 phage detection for wastewater RNA sample 26 . 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 July 16, 2021. ; https://doi.org/10.1101/2021.07.14.21260495 doi: medRxiv preprint (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 July 16, 2021. ; https://doi.org/10.1101/2021.07.14.21260495 doi: medRxiv preprint 6 repetitions and a standard deviation was calculated. Linear regression was performed between the log copy number and the Ct values from the RT-qPCR results. In order to validate the RT-qPCR designed assay's sensitivity in a wastewater matrix, RNA extracted from pre-determined negative wastewater sample for SARS-CoV-2 was examined using standard CDC's detection sets. The negative RNA sample was supplemented with known concentrations of a desired gene block (10 0 /10 1 /10 2 ). All experiments executed with the matrix pre-determined as negative for additional verification of the sample as negative. Eight repetitions were performed for each viral concentration or control. Ct results were plotted to represent the new probes limit of detection in a complex environment. gene contains a deletion known as Δ157-158. Accordingly, our designed primers-probe sets for variants detection focused on these regions and is presented in Figure 1 . 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 July 16, 2021. ; https://doi.org/10.1101/2021.07.14.21260495 doi: medRxiv preprint (Fig. 1a) . Using a detection set comprised of two primers meant to amplify the target region, a single probe (P.1 probe) was 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 July 16, 2021. ; https://doi.org/10.1101/2021.07.14.21260495 doi: medRxiv preprint 8 designed for the detection of the Gamma variant only (corresponding to the insertion). For Delta variant detection, the designated detection region was chosen from within the S gene. Focusing on S gene's 21989-22083 bp of the original sequence, the original SARS-CoV-2 sequence is identical to the Delta variant sequence with the exception of a 6 nucleotides deletion in the Delta lineage (Fig. 1a) . Using a detection set comprised of two primers meant to amplify the target region, a single probe (S∆157 probe) was designed for the detection of the Delta variant only (corresponding to the deletion). To ensure functionality, the described sets of primers and probes underwent characterization. Initially, a calibration curve was generated for the two primers-probe sets, using dsDNA as a template. A detection range of between 10 6 copies and 10 0 copies per µL was tested for each set (Fig. 1c) . Linear regression performed for the two probes demonstrated strong correlation. A limit of detection (LOD) could be determined for each primers-probe set and was identified as 10 0 copies per µL for the two sets ( Fig. 1d ), meaning highly sensitive detection. Specificity of the described sets was examined with either dsDNA fragments from the original SARS-CoV-2, relevant to the assigned detection regions, or using wastewater found positive for the original SARS-CoV-2 and the Alpha variant. Both sets did not manifest a signal when tested with such negative controls and were therefore found to be highly specific. Primers-probe sets were further characterized within a more intricate matrix. Wastewater from Binyamina, Israel served as a more complex environment for the RT-qPCR assay, as sewage samples contain a variety of materials and organisms. Wastewater samples were pre-determined as negative for SARS-CoV-2 using N gene detection. After confirming that the wastewater samples were negative, relevant dsDNA template copies were added exogenously at known concentrations (Fig. 2) . These samples were then used for detection by the examined primers-probe sets. As can be seen in Figure 2 , despite the wastewater matrix, P.1 and S∆157 probes maintained high functionality with a LOD of 10 0 copies per µL, proving their sensitivity and stability. 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 July 16, 2021. ; https://doi.org/10.1101/2021.07.14.21260495 doi: medRxiv preprint After fully characterizing the developed primers-probe sets, revealing their specificity, sensitivity and durability, it was possible to employ them on recently collected wastewater samples. Until recently, the Alpha variant was most dominant in Israel, however a noticeable decrease was observed in the past months. In the past month (May-June 2021), reports indicated that the Delta variant had reached Israel and is responsible for the majority of new morbidity cases. Knowing this, samples from different locations at the city of Modi'in were collected and tested for general SARS-CoV-2 presence, using N gene detection, and Alpha and Delta variants presence, using relevant S gene detection (Table 1 ). (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 July 16, 2021. ; https://doi.org/10.1101/2021.07.14.21260495 doi: medRxiv preprint As can be seen in Table 1 , all of the tested samples were positive to SARS-CoV-2 presence through N gene detection. However, none of the samples resulted in a positive detection for the Alpha variant, corresponding to the disappearance of this variant in clinical tests as reported by health officials. Furthermore, all samples, apart from one, resulted in a positive signal for the Delta variant using the designed primers-probe set described here. These field results indicated the new probe sets ability to correctly detect and quantify the Delta variant in a sensitive manner, even within a complex environment, and provide an assessment for variant of concern geographical spread in the population. Considering the importance of wastewater monitoring, as well as the possibility of clinical employment, we hope the developed sets can serve for variants of concern detection. SARS-CoV-2 variants, spike mutations and immune escape Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus SARS-CoV-2 D614G variant exhibits efficient replication ex vivo and transmission in vivo. Science (80-. ) Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England. 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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 Diagnosing the novel SARS-CoV-2 by quantitative RT-PCR: variations and opportunities Direct RT-qPCR assay for SARS-CoV-2 variants of concern (Alpha, B.1.1.7 and Beta, B.1.351) detection and quantification in wastewater Multiplex SARS-CoV-2 Genotyping RT-PCR for Population-Level Variant Screening and Epidemiologic Surveillance Tracking SARS-CoV-2 in Sewage: Evidence of Changes in Virus Variant Predominance during COVID-19 Pandemic Regressing SARS-CoV-2 sewage measurements onto COVID-19 burden in the population: A proof-of-concept for quantitative environmental surveillance First confirmed detection of SARS-CoV-2 in untreated wastewater in Australia: A proof of concept for the wastewater surveillance of COVID-19 in the community First detection of SARS-CoV-2 in untreated wastewaters in Italy Post-lockdown detection of SARS-CoV-2 RNA in the wastewater of Montpellier, France. One Heal Detection of SARS-CoV-2 in raw and treated wastewater in Germany -Suitability for COVID-19 surveillance and potential transmission risks SARS-CoV-2 Titers in Wastewater Are Higher than Expected from Clinically Confirmed Cases Use of bacteriophage MS2 as an internal control in viral reverse transcription-PCR assays We thank KANDO and the Israeli Ministry of Health for providing us with the sewage samples. We gratefully acknowledge GISAID database for access to SARS-CoV-2 variants sequences. We gratefully acknowledge Esti Kramarsky-Winter assistance for comments and scientific editing of the manuscript. 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 July 16, 2021. ; https://doi.org/10.1101/2021.07.14.21260495 doi: medRxiv preprint 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 July 16, 2021. ; https://doi.org/10.1101/2021.07.14.21260495 doi: medRxiv preprint