RU2795014C1 - OLIGONUCLEOTIDES FOR DETECTION OF SARS-CoV-70 MUTATION S:delHV69 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: oligonucleotides for detection of SARS-CoV-2 mutation S:delHV69-70, forward primer d6Acv-F, reverse primer d6Acv-R, fluorescent probe d6Ocv-Z1Y, are presented by unique sequences SEQ ID NO: 1-3 respectively. At the same time, to create forward and reverse primers and a fluorescent probe, use fragments of the reference genomes of SARS-CoV-2 wild type, Alpha (В. 1.1.7), Omicron (В.1.1.529), Eta (В.1.525), В. 1.620, С.36.3. Synthesized oligonucleotides SEQ ID NO NO: 1-3 do not form cross-reactions with other tested samples, selected site is amplified with 100% specificity allowing to determine presence or absence of a significant target S:delHV69 in the samples of biological material.

EFFECT: determination of SARS-CoV-2 mutation S:delHV69-70.

3 cl, 1 tbl, 5 ex

Description

The invention relates to the field of biotechnology, in particular to the determination of the SARS-CoV-2 S:delHV69-70 mutation using real-time polymerase chain reaction (RT-PCR) and can be used to identify S ARS-CoV-2 genovariants in epidemiological studies .

Coronavirus infection (COVID-19) is an infectious disease caused by SARS-CoV-2. The virus is transmitted from person to person by airborne droplets. Changes in the structure of the SARS-CoV-2 genome lead to the emergence of variants that WHO has designated as of interest (VOIs, Variants of Interest), of concern (VOCs, Variants of Concern) and VOC lineages under monitoring (VOC- LUM).Determination of mutations in the SARS-CoV-2 genome and their classification into VOIs and VOCs and VOC-LUM is an important element of molecular genetic monitoring of strains of a new coronavirus infection. Mutations, the detection of which is necessary, are listed in the reports of the Technical Advisory Group on the Evolution of the Virus SARS-CoV-2 (TAG-VE) WHO [https://www.who.int/publications/m/item/terms-of-reference-for-the-technicsl-advisory-group-on-sars-cov- 2-virus-evolution-(tag-ve)].

To determine virus genovariants and conduct molecular genetic monitoring of SARS-CoV-2, whole genome and fragment sequencing methods are used, which are expensive, time-consuming methods. In order to detect SARS-CoV-2, the World Health Organization proposed isolation of the causative agent RNA followed by reverse transcription polymerase chain reaction (RT-PCR) [https://www.who.int/emergencies/diseases/novel-coronavirus-2019/ technical-guidance/laboratory-guidance].

Known research protocols are aimed at identifying specific nucleotide sequences in genes and intergenic spaces:

- ORF1ab, N [http://ivdc.chinacdc.cn/kyjz/202001/t20200121_211337.html];

- RdRP, E, N [https://www.who.int/docs/default-source/coronaviruse/protocol-v2-1.pdf?sfvrsn=a9ef618c_2];

- ORF1b-nsp14, N [https://www.who.int/docs/default-source/coronaviruse/peiris-protocol-16-l-20.pdf?sfvrsn=af1aac73_4];

- S, N [https://www.who.int/docs/default-source/coronaviruse/method-niid-20200123-2.pdf?sfvrsn=fbf75320_7];

- N [https://www.who.int/docs/default-source/coronaviruse/conventional-rt-pcr-followed-by-sequencing-for-detection-of-ncov-rirl-nat-inst-health-t .pdf?sfvrsn=42271c6d_4];

-N

[https://www.fda.gov/media/134922/download, https://www.who.int/docs/default-source/coronaviruse/uscdcrt-pcr-panel-primer-probes.pdf?sfvrsn=fa29cb4b_2 ];

- RdRP [https://www.who.int/docs/default-source/coronaviruse/real-time-rt-pcr-assays-for-the-detection-of-sars-cov-2-institut-pasteur-paris .pdf?sfvrsn=3662fcb6 2].

At the same time, the analysis of genome-wide nucleotide sequences of SARS-CoV-2 showed the frequent presence of mutations (especially deletions) in the genomes of this coronavirus. In this regard, the main disadvantage of the above analogs is the risk of obtaining false negative RT-PCR results due to blocking the reaction in the presence of mutations in the region of the amplified region.

A screening strategy for identifying the dominant variant of S ARS-COV-2 in the fifth peak of Kurdistan-Iran population using HRM and Probe-based RT-PCR assay is known from the prior art. . Mohammad Moradzadac, Hasan Soltani, Hamid Salehi, Khaled Rahmani, Dariush Khateri, Mohammad Zaid Rahimi, Diman Az, Shohreh Fakhari, Journal of Virological Methods, Volume 304, June 2022, 114514, https://doi.org/10.1016 j.jviromet .2022.114514]. where the detection of the S protein mutation S:delHV69-70 is carried out using a high resolution melting assay.

Multiplex kits of reagents for the detection of genovariants of the SARS-CoV-2 virus by PCR have been developed by several manufacturing companies. The method proposed by ThermoFisher Scientific is based on the use of TaqMan type probes to detect several mutations at once, specific for different genovariants [SARS-CoV-2 Variants Detection Using TaqMan SARS-CoV-2 Mutation Panel Molecular Genotyping Assays. Puja Neopane, Jerome Nypaver, Rojeet Shrestha, Safedin Sajo Beqaj, https://doi.org/10.2147/IDR.S3355831. Kits that provide multiplex RT-PCR after reverse transcription are user-friendly [1) Two-step strategy for the identification of SARS-CoV-2 variant of concern 202012/01 and other variants with spike deletion H69-V70, France , August to December 2020. Antonin Bal, Gregory Destras, Alexandre Gaymard, Karl Stefic, Julien Marlet, Sebastien Eymieux, Hadrien Regue, Quentin Semanas, Constance d'Aubarede, Genevieve Billaud, Frederic Laurent, Claudia Gonzalez, Yahia Mekki, Martine Valette, Maude Bouscambert, Catherine Gaudy-Graffin, Bruno Lina, Florence Morfin, Laurence Josset, Jean-Sebastien Casalegno, Emilie Frobert, Vanessa Escuret, Vinca Icard, Marion Jeannoel, Marie-Paule Milon, Christophe Ramiere, Caroline Scholtes, Jean-Claude Tardy, Mary-Anne Trabaud, Isabelle Schuffenecker, DOI: 10.2807/1560-7917.ES.2021.26.3.2100008; 2) Recurrent emergence of SARS-CoV-2 spike deletion H69/V70 and its role in the Alpha variant B. 1.1.7. Bo Meng, Steven A Kemp, Guido Papa, Rawlings Datir, Isabella ATM Ferreira, Sara Marelli, William T Harvey, Spyros Lytras, Ahmed Mohamed, Giulia Gallo, Nazia Thakur, Dami A Collier, Petra Mlcochova, COVID-19 Genomics UK (COG -UK) Consortium, Lidia M Duncan, Alessandro M Carabelli, Julia C Kenyon, Andrew M Lever, Anna De Marco, Christian Saliba, Katja Culap, Elisabetta Cameroni, Nicholas J Matheson, Luca Piccoli, Davide Corti, Leo C James, David L Robertson, Dalan Bailey, Ravindra K Gupta, DOI: 10.1016/j.celrep.2021.109292; 3) Whole-genome sequencing of SARS-CoV-2 showed a wide spread of B. 1.525 in February 2021 in Libya. Inas M Alhudiri, Ahmad M Ramadan, Khaled M Ibrahim, Adel Abdalla, Mouna Eljilani, Mohamed Ali Salem, Hajer Mohamed Elgheriani, Salah Edin El Meshri, Adam Elzagheid, https://doi.org/10.1080/19932820.2021.2001210; 4) The challenge of screening SARS-CoV-2 variants of concern with RT-qPCR: One variant can hide another. Jose Valter Joaquim Silva Junior, higryd Mercioratto, Pablo Sebastian Britto de Oliveira, Thaisa Regina Rocha Lopes, Patricia Chaves Brites, Elehu Moura de Oliveira, Rudi Weiblen, Eduardo Furtado Flores, https://doi.org/10.1016/j.jviromet. 2021.114248; 5) Rapid and simultaneous identification of three mutations by the Novaplex™ SARS-CoV-2 variants I assay kit. Wakaki Kami, Takeshi Kinjo, Wakako Arakaki, Hiroya Oki, Daisuke Motooka, Shota Nakamura, Jiro Fujita, https://doi.org/10.1016/j.jcv.2021.104877; 6) Patent CN 113817868, 07/08/2021, 12/21/2021; 7) Patent CN 113215312, 04/28/2021, 08/06/2021; 8) Patent CN 113005226, 02/07/2021, 06/22/2021.]. However, the use of these kits does not imply a quick possibility of replacing targets for the detection of new genovariants containing missense mutations or substitutions.

The detection of additional targets specific for emerging genovariants, as well as the exclusion of targets for circulating genovariants from the analysis, makes it possible to increase the efficiency of laboratory research as a whole. In this regard, there is a need to develop oligonucleotides to detect the S:delHV69-70 mutation in biological samples with the confirmed presence of SARS-CoV-2 RNA: primers and a fluorescently labeled probe for real-time PCR.

The technical result of the claimed invention is aimed at detecting the S:delHV69-70 mutation in biological samples with a confirmed presence of SARS-CoV-2 RNA with a high degree of specificity through such oligonucleotide primers and a fluorescently labeled probe that allow you to effectively determine the genovariant S ARS-CoV- 2 using widely available techniques and materials.

The technical result is achieved through the use of chemically synthesized oligonucleotides to determine the presence of the S:delHV69-70 mutation in a biological sample with a confirmed presence of SARS-CoV-2 RNA, having the following oligonucleotide composition:

forward primer d6Acv-F - SEQ ID NO: 1;

reverse primer d6Acv-R - SEQ ID NO: 2;

fluorescent probe d60cv-Z1Y - SEQ ID NO: 3.

Primers are oligonucleotide sequences for amplification of a fragment containing the S:delHV69-70 mutation in biological samples with confirmed presence of SARS-CoV-2 RNA, a fluorescently labeled conformationally blocked probe is an oligonucleotide containing a fluorophore and a fluorescence quencher that allows the detection of the amplified fragment.

The claimed oligonucleotides were developed on the basis of the known sequence of the SARS-CoV-2 S gene, taken from the NCBI database (https://www.ncbi.nlm.nih.gov/), as a result of which a fragment was selected for the detection of the mutation of the S gene S :delHV69-70. Primers and a probe for amplification of 133/127 base pairs were selected for the selected fragment: direct primer d6Acv-F - SEQ ID NO: 1; reverse primer d6Acv-R - SEQ ID NO: 2 and fluorescent probe d60cv-Z1Y - SEQ ID NO: 3.

The claimed invention is the result of work in the framework of improving the molecular genetic monitoring of SARS-CoV-2 variants, carried out at the Central Research Institute of Epidemiology of Rospotrebnadzor (Moscow, Russia).

To select target sequences - oligonucleotide landing sites, use fragments of reference genomes taken from the NCBI database (https://www.ncbi.nlm.nih.gov/), both wild-type SARS-CoV-2 and those genovariants, in which the S:delHV69-70 mutation occurs (Alpha (B.1.1.7), Omicron (B.1.1.529), Eta (B.1.525), B.1.620, C.36.3.). To search for conservative sequences, modern algorithms for in silico analysis of nucleotide sequences and programs that are in the public domain, including AlignX, SnapGene Viewer, MEGA, Unipro UGENE, are used. Compile a list of significant mutations characteristic of genovariants. Then, the oligonucleotide sequences of the forward and reverse primers, as well as the fluorescently labeled probe, are selected. For detection of samples containing the mutation S:delHV69-70, use the channel for the detection of the fluorophore R6G. Mentioned oligonucleotide sequences are shown in Table 1.

Analysis of the claimed sequences using the Primer BLAST resource demonstrated that the forward primer d6Acv-F (SEQ ID NO: 1) and the reverse primer d6Acv-R (SEQ ID NO: 2) amplify the region in which the S:delHV69-70 mutation is located with 100 % specificity.

Nasopharyngeal and oropharyngeal swabs/exudates are used as biological material, with confirmed presence of SARS-CoV-2 RNA (for example, after analysis on the AmpliSense® COVTD-19-FL reagent kit).

Isolation of RNA from biological material is carried out in accordance with MU 1.3.2569-09 "Organization of the work of laboratories using nucleic acid amplification methods when working with material containing microorganisms of I-IV pathogenicity groups." Isolation of RNA from biological material is carried out using a kit of reagents in accordance with the manufacturer's instructions. For RNA isolation, the RIBO-prep reagent kit (Federal Scientific Research Institute of Epidemiology of Rospotrebnadzor, Russia) or any similar kit for RNA isolation can be used. The optimal concentration of RNA is 10 ³ - 10 ⁵ copies per 10 µl. The reverse transcription (RT) reaction is carried out using a kit of reagents in accordance with the manufacturer's instructions. To carry out the OT reaction, the REVERTA-L reagent kit (Federal Scientific Research Institute of Epidemiology of Rospotrebnadzor, Moscow, Russia) or any similar kit can be used. The finished cDNA preparation can be stored at a temperature of 2 to 8°C for a week, at a temperature of minus 24 to minus 16°C for 6 months and at a temperature not exceeding minus 68°C for a year.

Polymerase chain reaction (PCR) is an efficient method for obtaining in vitro a large number of copies of specific nucleotide sequences. Their amplification is carried out in a three-step cyclical process.

The process of PCR amplification consists in repeated repetition of the processes of denaturation, renaturation and synthesis. Denaturation (95°C) - thermal effect on the DNA molecule in order to obtain a single-stranded structure. Renature (55-60°C)-primers added to the reaction pair with separated strands. Synthesis (70-75°C) - synthesis of the second strand of DNA. Each cycle lasts 3-5 minutes.

Data analysis is carried out on the basis of detection by the amplifier of the level of the fluorescent signal emitted by the intercalating dye. With an increase in the number of copies of the analyzed area, an exponential growth of the fluorescent signal is detected. As a result, an S-shaped curve is observed in the case of a signal specific to the fluorescent probe, or its absence in the case of a non-probe-specific sequence. Analysis of the curves makes it possible to judge the absence or presence of a mutation in the studied samples.

PCR-RT is carried out using the claimed, presented in Table 1, oligonucleotides - primers and probe, for the detection of mutations S:delHV69-70 SARS-CoV-2.

PCR-RT is carried out under the following conditions:

The total volume of the reaction mixture is 25 μl.

PCR components are mixed as follows:

(a) 10 µl of a mixture containing:

- oligonucleotide primers SEQ ID NO NO: 1, 2 - 0.7 mm each;

- fluorescent probe SEQ ID NO: 3 - 0.2 mm;

- dNTPs - 0.44 mM.

(b) a reagent containing a recombinant Taq DNA polymerase enzyme, for example, 0.5 µl TaqF Polymerase (Central Research Institute of Epidemiology of Rospotrebnadzor, Moscow, Russia) or any similar commercial kit in accordance with the manufacturer's instructions.

(c) PCR buffer containing MgCl ₂ , for example, 5.0 µl of PCR buffer "RT-PCR-mixture-2 FEP/FRT" (Federal Budgetary Institution of Central Research Institute of Epidemiology of Rospotrebnadzor, Moscow, Russia) or any similar commercial kit in accordance with manufacturer's instructions.

(d) cDNA obtained by reverse transcription - 10 µl.

Amplification is carried out on a device with the possibility of fluorescence detection, for example, "Rotor-Gene Q" ("Qiagen", Germany) or on any other device with 2-5 detection channels in accordance with the manufacturer's instructions.

Amplification is carried out according to the following program: 1 cycle 95°C for 15 minutes, 45 cycles at 95°C - 10 seconds / 60°C - 20 seconds. Fluorescence detection is carried out at the 60°C stage through the R6G fluorophore channel.

To analyze the results, a threshold line is set corresponding to a value of 10% of the average fluorescence signal of a sample with the S:delHV69-70 mutation. Samples for which the fluorescence curves cross the threshold line, and the kinetics of accumulation of the fluorescent signal is exponential, are positive, that is, they contain the SARS-CoV-2 S:delHV69-70 mutation.

The implementation of the claimed invention is illustrated by the following examples:

Example 1 Preparation of oligonucleotides to detect the S:delHV69-70 mutation of SARS-CoV-2.

For the selection of target sequences - oligonucleotide landing sites, fragments of reference genomes from the NCBI database (https://www.ncbi.nlm.nih.gov/) of wild-type SARS-CoV-2 and Alpha genovariants (B. 1.1.7) were used , refseq MZ344997 (NCBI); Omicron (V. 1.1.529), refseq OL672836 (NCBI). To search for mutations, modern algorithms for in silico analysis of nucleotide sequences and programs that are in the public domain, including Unipro UGENE, Oligo Calculators (Oligo Calculators) Integrated DNA Technologies, Inc., were used. (Oligo Analyzer (idtdna.com)). The section of the S gene, which is characteristic only for the S:delHV69-70 mutation of SARS-CoV-2, was selected, to which the oligonucleotide sequences of the forward d6Acv-F and reverse d6Acv-R primers, as well as the fluorescent probe d60cv-Z1Y, were selected.

Analysis of these sequences using the Primer BLAST resource showed that the d6Acv-F forward and d6Acv-R reverse primers amplify the S:delHV69-70 mutation site with 100% specificity.

Oligonucleotides for detection of mutation S:delHV69-70 SARS-CoV-2 - forward primer d6Acv-F, reverse primer d6Acv-R, fluorescent probe d6Ocv-Z1Y, are represented by unique sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively.

Example 2 Detection of the S:delHV69-70 mutation in SARS-CoV-2 by RT-PCR.

The determination of the SARS-CoV-2 S:delHV69-70 mutation is carried out by real-time PCR under the following conditions:

The total volume of the reaction mixture is 25 μl.

PCR components are mixed as follows:

(a) 10 µl of a mixture containing:

- oligonucleotide primers: SEQ ID NO: 1, SEQ ID NO: 2 - no 0.7 mm;

- fluorescent probe: SEQ ID NO: 3 - 0.2 mm;

- dNTPs - 0.44 mM.

(b) 0.5 µl of TaqF Polymerase reagent (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) containing the recombinant Taq DNA polymerase enzyme.

(c) 5.0 μl of PCR buffer "RT-PCR-mixture-2 FEP/FRT" (Federal Budgetary Institution of Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) containing MgCl2.

(d) cDNA obtained after the reverse transcription reaction (REVERTA-L, Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) - 10 µl.

Real-time PCR was performed with fluorescent detection on a Rotor-Gene Q instrument with 5 detection channels (Qiagen, Germany).

The material prepared by the described method, containing unique oligonucleotide sequences of SEQ ID NO: 1-3, is used to determine the SARS-CoV-2 S:delHV69-70 mutation in biological material samples.

Example 3 Detection of SARS-CoV-2 S:delHV69-70 mutation in biological samples.

To determine the S:delHV69-70 SARS-CoV-2 mutation, 88 samples were selected, with the confirmed presence of SARS-CoV-2 RNA.

For the study, clinical material was used - swabs / discharge of the nasopharynx and oropharynx containing RNA S ARS-CoV-2. The presence of RNA virus S ARS-CoV-2 was confirmed by laboratory using the AmpliSense® COVID-19-FL reagent kit (Federal Scientific Research Institute of Epidemiology of Rospotrebnadzor, Moscow, Russia).

Isolation of RNA from biological material was carried out in accordance with MU 1.3.2569-09 "Organization of the work of laboratories using nucleic acid amplification methods when working with material containing microorganisms of pathogenicity groups I-IV". RNA was isolated by nucleoprecipitation using the RIBO-prep reagent kit (Central Research Institute of Epidemiology of Rospotrebnadzor, Moscow, Russia) in accordance with the instructions for the kit. Reverse transcription was performed using the REVERTA-L reagent kit (Central Research Institute of Epidemiology of Rospotrebnadzor, Moscow, Russia) in accordance with the instructions for the kit.

The presence of SARS-CoV-2 RNA was laboratory confirmed using the AmpliSense® COVID-19-FL reagent kit (Central Research Institute of Epidemiology of Rospotrebnadzor, Moscow, Russia). The sensitivity of this kit in relation to swabs/detachable nasopharynx and oropharynx, according to the instructions, is 1*10 ⁴ . Specificity - 100%. RT-PCR was performed under the conditions described in Example 2 using unique oligonucleotide sequences of SEQ ID NOs: 1-3.

Amplification was carried out on the device "DT-96" ("DNA-Technology", Russia) according to the following program: 1 cycle 95°C for 15 minutes, 45 cycles at a temperature of 95°C -10 seconds / 60°C - 20 seconds. Fluorescence detection was carried out at the 60°C stage through the R6G fluorophore channel. To analyze the results, a threshold line was set corresponding to a value of 10% of the average fluorescence signal of a sample with the S:delHV69-70 mutation. For 87 samples, the fluorescence curve crossed the threshold line and, at the same time, the kinetics of accumulation of the fluorescent signal was exponential, indicating that these samples contain the SARS-CoV-2 S:delHV69-70 mutation.

For these 87 samples, SARS-CoV-2 S:delHV69-70 mutation was confirmed by fragment sequencing using the Sanger sequencing method. Fragment sequencing was performed on an ABI 3500xL genetic analyzer (Applied Biosystems, USA), alignment and analysis of the obtained sequences was performed using the AlignX program (Thermo Fisher Scientific, USA). The absence of mutation in 1 sample without a fluorescent signal was also confirmed by fragment sequencing.

Thus, 87 samples containing the S:delHV69-70 SARS-CoV-2 mutation were identified from the entire sample.

Example 4. Detection of SARS-CoV-2 mutation S:delHV69-70 in biological samples.

To determine the SARS-CoV-2 S:delHV69-70 mutation, 96 samples were selected, with the confirmed presence of SARS-CoV-2 RNA.

For the study, clinical material was used - swabs / discharge of the nasopharynx and oropharynx containing SARS-CoV-2 RNA.

Isolation of RNA from biological material was carried out in accordance with MU 1.3.2569-09 "Organization of the work of laboratories using nucleic acid amplification methods when working with material containing microorganisms of pathogenicity groups I-IV". RNA was isolated by nucleoprecipitation using the RIBO-prep reagent kit (Central Research Institute of Epidemiology of Rospotrebnadzor, Moscow, Russia) in accordance with the instructions for the kit. Reverse transcription was performed using the REVERTA-L reagent kit (Central Research Institute of Epidemiology of Rospotrebnadzor, Moscow, Russia) in accordance with the instructions for the kit. The presence of SARS-CoV-2 virus RNA was laboratory confirmed using the AmpliSense® COVID-19-FL reagent kit (Central Research Institute of Epidemiology of Rospotrebnadzor, Moscow, Russia). The sensitivity of this kit in relation to swabs / detachable nasopharynx and oropharynx, according to the instructions, is 1 * 10 ⁴ copies / ml. Specificity - 100%.

RT-PCR was performed under the conditions described in Example 2 using unique oligonucleotide sequences of SEQ ID NOs: 1-3.

Amplification was carried out on a Real-time CFX96 Touch device (Bio-Rad, USA) according to the following program: 1 cycle at 95°C for 15 minutes, 45 cycles at 95°C for 10 seconds / 60°C for 20 seconds. Fluorescence detection was carried out at the 60°C stage through the R6G fluorophore channel. To analyze the results, a threshold line was set corresponding to a value of 10% of the average fluorescence signal of a sample with the S:delHV69-70 mutation. For 71 samples, the fluorescence curve crossed the threshold line and, at the same time, the kinetics of accumulation of the fluorescent signal was exponential, which indicates that these samples contain the SARS-CoV-2 S:delHV69-70 mutation. The absence of mutation in 25 samples without a fluorescent signal was also confirmed by fragment sequencing.

Thus, 71 samples containing the S:delHV69-70 SARS-CoV-2 mutation were identified from the entire sample.

Example 5 Detection of SARS-CoV-2 mutation S:delHV69-70 in biological samples.

To determine the SARS-CoV-2 S:delHV69-70 mutation, 5 samples were selected, with the confirmed presence of SARS-CoV-2 RNA.

For the study, clinical material was used - swabs / discharge of the nasopharynx and oropharynx containing SARS-Cov-2 RNA. Isolation of RNA from biological material was carried out in accordance with MU 1.3.2569-09 "Organization of work of laboratories using nucleic acid amplification methods when working with material containing microorganisms of pathogenicity groups I-IV". RNA isolation was carried out by nucleoprecipitation using the RIBO-prep reagent kit (Central Research Institute of Epidemiology of Rospotrebnadzor, Moscow, Russia) in accordance with the instructions for the kit. Reverse transcription was performed using the REVERTA-L reagent kit (Central Research Institute of Epidemiology of Rospotrebnadzor, Moscow, Russia) in accordance with the instructions for the kit. The presence of SARS-CoV-2 RNA was laboratory confirmed using the AmpliSense® COVID-19-FL reagent kit (Central Research Institute of Epidemiology of Rospotrebnadzor, Moscow, Russia). The sensitivity of this kit in relation to smears / detachable nasopharynx and oropharynx, according to the instructions, is 1*10 ⁴ . Specificity - 100%.

RT-PCR was performed under the conditions described in Example 2 using unique oligonucleotide sequences of SEQ ID NOs: 1-3.

Amplification was carried out on a Rotor-Gene Q device (Qiagen, Germany) according to the following program: 1 cycle at 95°C for 15 minutes, 45 cycles at 95°C -10 seconds / 60°C - 20 seconds. Fluorescence detection was carried out at the 60°C stage through the R6G fluorophore channel. To analyze the results, a threshold line was set corresponding to a value of 10% of the average fluorescence signal of a sample with the S:delHV69-70 mutation. The fluorescence curve did not cross the threshold line, which indicated that there were no samples containing the S:delHV69-70 mutation of the SARS-CoV-2 virus in the sample.

The presence of other mutations of the SARS-CoV-2 virus in these 5 samples and the absence of the S:delHV69-70 mutation was confirmed by fragment sequencing using the Sanger sequencing method. Fragment sequencing was performed on an ABI 3500xL genetic analyzer (Applied Biosystems, USA), alignment and analysis of the resulting sequences were performed using the AlignX program (Thermo Fisher Scientific), USA).

The claimed invention makes it possible to detect the S:delHV69-70 mutation of SARS-CoV-2 in biological samples with confirmed presence of SARS-CoV-2 virus RNA. Synthesized oligonucleotides SEQ ID NO NO: 1-3 do not give cross-reactions with other tested samples, amplify a given site with 100% specificity and allow you to determine the presence or absence of a significant target S:delHV69-70 in the samples of biological material.

Claims (6)

1. A set of primers and a probe for determining the SARS-CoV-2 S:delHV69-70 mutation, having the following composition: forward primer d6Acv-F - SEQ ID NO: 1; reverse primer d6Acv-R - SEQ ID NO: 2; fluorescent probe d6Ocv-Z1Y - SEQ ID NO: 3. 2. A set of primers and a probe for determining the SARS-CoV-2 S:delHV69-70 mutation according to claim 1, where to create forward and reverse primers, a fluorescent probe, fragments of the SARS-CoV-2 reference genomes of the wild type, Alpha (B. 1.1.7), Omicron (B.1.1.529), Eta (B.1.525), B.1.620, C.36.3. 3. A set of primers and a probe for detecting the SARS-CoV-2 S:delHV69-70 mutation according to claim 1, where the fluorescent probe is an oligonucleotide containing the R6G fluorophore and the BHQ1 fluorescence quencher, which allows the amplified fragment to be detected.