RU2791958C1 - OLIGONUCLEOTIDES FOR DETECTION OF SARS-CoV-2 MUTATION S:N501Y - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, to determination of the SARS-CoV-2 S:N501Y mutation by real-time polymerase chain reaction. Oligonucleotides for detection of SARS-CoV-2 mutation S:N501Y are described: forward primer 501Ocv-F2, reverse primer 1501cv-R, fluorescent probe 1501cv-Z, represented by unique sequences SEQ ID NO: 1-3 respectively. At the same time, to create forward and reverse primers, a fluorescent probe, fragments of the reference genomes of SARS-CoV-2 wild type, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Omicron (B. 1.1.529), Mu (B.1.621), Theta (P.3), (B.1.640), (C.1.2) are used. Synthesized oligonucleotides SEQ ID NO NO: 1-3 do not form cross-reactions with other tested samples, a selected site is amplified with 100% specificity allowing to determine presence or absence of a significant target S:N501Y in the samples of biological material.

EFFECT: determining of SARS-CoV-2 S:N501Y mutation by real-time polymerase chain reaction.

1 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:N501Y mutation using real-time polymerase chain reaction (RT-PCR) and can be used to identify SARS-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/rn 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 and laborious. 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-16-1-20.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 combination of a primer and a probe for detecting mutant strains of a new coronavirus is known from the prior art, which contains primers with specific nucleotide sequences and probes with specific nucleotide sequences. Based on the combination of primer and probe, the invention establishes a method for detecting the N501Y mutation site of the N gene and the D614G mutation site of the N gene of a new coronavirus based on nanoparticle PCR technology [Patent CN113278733, filed on 05/21/2021, published on 08/20/2021]. A significant disadvantage of this solution is the complication of the stages of sample preparation, in addition, the method requires additional costs to provide the system with nanoparticles.

Kits that provide multiplex RT-PCR after reverse transcription are user-friendly [1) The challenge of screening SARS-CoV-2 variants of concern with RT-qPCR: One variant can hide another. Jose Valter Joaquim Silva Junior, mgryd 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; 2) 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/i.icv.2021.104877; 3) Patent RU 2761025, 07/26/2021, 12/02/2021 4) Patent CN113817868, 07/08/2021, 12/21/2021; 5) Patent CN113215312, 04/28/2021, 08/06/2021; 6) Patent CN113005226, 02/07/2021, 06/22/2021; 7) Patent CN113278733, 05/21/2021, 08/20/2021]. 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 identify the S:N501 Y mutation as unique and significant in the evolution of the virus in biological samples with confirmed presence of SARS-CoV-2 RNA. The development is carried out by selecting primers and a fluorescently labeled probe for real-time PCR.

The technical result of the claimed invention is aimed at detecting the S:N501Y mutation in biological samples with the confirmed presence of SARS-CoV-2 RNA with a high degree of specificity by means of primer oligonucleotides and a fluorescently labeled probe, which make it possible to effectively determine the S:N501Y mutation of SARS-CoV-2 with 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:N501Y mutation in a biological sample with a confirmed presence of SARS-CoV-2 RNA, having the following oligonucleotide composition:

forward primer 501Ocv-F2 - SEQ ID NO: 1;

reverse primer 1501cv-R - SEQ ID NO: 2;

fluorescent probe 1501cv-Z - SEQ ID NO: 3.

Primers are oligonucleotide sequences for amplification of a fragment containing the S:N501Y 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 an amplified fragment.

The claimed oligonucleotides were developed on the basis of the analysis of the SARS-CoV-2 S gene sequences 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 S mutation: N501Y. Primers and a probe for amplification of 114 base pairs were selected for the selected fragment: forward primer 501Ocv-F2 - SEQ ID NO: 1; reverse primer 1501cv-R - SEQ ID NO: 2 and fluorescent probe 1501cv-Z - SEQ ID NO: 3.

The claimed invention is the result of work in the framework of improving the molecular genetic monitoring of variants of the SARS-CoV-2 virus, 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:N501Y mutation occurs: Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Omicron (B.1.1.529), Mu (B.1.621), Theta (P .3), (B.1.640), (C.1.2). To search for conservative regions of 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. To detect samples containing the S:N501Y mutation, a FAM fluorophore detection channel is used. Mentioned oligonucleotide sequences are shown in Table 1.

Analysis of the claimed sequences using the Primer BLAST resource demonstrated that the forward primer 501Ocv-F2 (SEQ ID NO:l) and the reverse primer 1501cv-R (SEQ ID NO:2) amplify the site in which the S:N501Y mutation is located with 100% specificity .

Where FAM is a fluorophore, BHQ1 is a fluorescence quencher, + in front of the nucleotide is an LNA modification.

Nasopharyngeal and oropharyngeal swabs/sweeps are used as biological material, with confirmed presence of SARS-CoV-2 RNA (for example, after analysis on the AmpliSense ^® COVID-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. Renaturation (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:N501Y 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: 1, SEQ ID NO: 2 - 0.5 mM each;

- fluorescent probe SEQ ID NO: 3-0.15 mm;

- dNTPs - 0.44 mM.

(b) a reagent containing a recombinant enzyme Taq DNA polymerase, for example, 0.5 µl TaqF polymerase (Federal Budgetary Institution of 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 FAM 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:N501Y 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:N501Y mutation.

The implementation of the claimed invention is illustrated by the following examples: Example 1. Obtaining oligonucleotides to determine the mutation S:N501Y 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 both wild-type SARS-CoV-2 and those genovariants in which mutation S:N501Y (Alpha (B.1.1.7), refseq MZ344997 (NCBI); Beta (B.1.1.351), refseq MW598419 (NCBI); Gamma (P.1), refseq MZ169911 (NCBI); Omicron ( B.1.1.529), refseq OL672836 (NCBI) and others). To search for mutations, modern algorithms for in silico analysis of nucleotide sequences and programs that are in the public domain, including AlignX, Unipro UGENE, Oligo Calculators (Oligo Calculators) Integrated DNA Technologies, Inc., were used. (Oligo Analyzer (idtdna.com)). A list of conserved regions, characteristic only of the SARS-CoV-2 S:N501Y mutation, was compiled, to which the oligonucleotide sequences of the forward 501Ocv-F2 and reverse 1501cv-R primers, as well as the 1501cv-Z fluorescent probe, were selected.

Analysis of these sequences using the Primer BLAST resource showed that the forward 501Ocv-F2 and reverse 1501cv-R primers amplify the region in which the S:N501Y mutation is located with 100% specificity.

Oligonucleotides for detection of mutation S:N501Y SARS-CoV-2 - forward primer 501Ocv-F2, reverse primer 1501cv-R, fluorescent probe 1501cv-Z, 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:N501Y mutation of SARS-CoV-2 by RT-PCR.

The determination of the S:N501Y mutation of SARS-CoV-2 is carried out by PCR-RT 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, 0.5 mM each;

- fluorescent probe: SEQ ID NO: 3-0.15 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" (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) containing MgCl ₂ .

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

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 S:N501Y mutation of SARS-CoV-2 in samples of biological material.

Example 3 Detection of SARS-CoV-2 mutation S:N501Y in biological samples.

To determine the SARS-CoV-2 S:N501Y 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 RNA of the SARS-CoV-2 virus. 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).

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.

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 channel for the FAM fluorophore. 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:N501Y mutation. For 94 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:N501Y mutation.

For these 94 samples, SARS-CoV-2 S:N501Y 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 absence of mutation in 2 samples without fluorescent signal was also confirmed by fragment sequencing.

Thus, 94 samples containing the S:N501Y SARS-CoV-2 mutation were identified from the entire sample.

Example 4. Detection of SARS-CoV-2 mutation S:N501Y in biological samples.

To determine the S:N501Y SARS-CoV-2 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. The presence of SARS-CoV-2 RNA was laboratory confirmed using the AmpliSense ^® COVTD-19-FL reagent kit (Central 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.

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 channel for the FAM fluorophore. 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:N501Y mutation. For 4 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:N501Y mutation.

For these 4 samples, SARS-CoV-2 S:N501Y 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 absence of mutation in 1 sample without a fluorescent signal was also confirmed by fragment sequencing.

Thus, 4 samples containing the S:N501Y SARS-CoV-2 mutation were identified from the entire sample.

Example 5 Detection of SARS-CoV-2 mutation S:N501Y in biological samples.

To determine the S:N501Y SARS-CoV-2 mutation, 15 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. The presence of SARS-CoV-2 RNA was laboratory confirmed using the AmpliSense ^® COVTD-19-FL reagent kit (Central 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.

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 channel for the FAM fluorophore. 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:N501Y mutation. The fluorescence curve did not cross the threshold line, indicating that there were no samples containing the SARS-CoV-2 mutation S:N501Y in the sample.

The presence of other SARS-CoV-2 mutations in these 15 samples, as well as the absence of the S:N501Y 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:N501Y mutation of SARS-CoV-2 in biological samples with confirmed presence of SARS-CoV-2 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:N501Y in the samples of biological material.

Claims (4)

A set of primers and a probe for determining the S:N501Y mutation of SARS-CoV-2, having the following composition: forward primer 501Ocv-F2 - SEQ ID NO: 1; reverse primer 1501cv-R - SEQ ID NO: 2; fluorescent probe 1501cv-Z - SEQ ID NO: 3.