RU2795017C1 - OLIGONUCLEOTIDES FOR DETECTION OF SARS-COV-2 MUTATION S:Ins214EPE - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: set of primers and a probe for determination of SARS-CoV-214 mutation S:Ins214EPE is described with the following composition: forward primer 214-F - SEQ ID NO: 1; reverse primer 214pure-R - SEQ ID NO: 2; fluorescent probe 214-Z2 - SEQ ID NO: 3. 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:Ins214EPE in the samples of biological material.

EFFECT: determination of SARS-CoV-2 mutation S:Ins214EPE.

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:Ins214EPE 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/m 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:

- ORFlab, 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=a9ef618 c_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 test system for the detection of SARS-CoV-2 of the Omicron line by the method of one-step reverse transcription polymerase chain reaction is known from the prior art [Patent RU 2772362, filed on 12/31/2021]. The specified test system includes oligonucleotide primers and a fluorescent probe with the following structure: forward primer Ins214EPESARS-CoV-2; reverse primer Ins214EPESARS-CoV-2; fluorescent probe Ins214EPESARS-CoV-2.

Multiplex reagent kits for the detection of SARS-CoV-2 genovariants 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 [Identifying SARS-CoV-2 Variants of Concern through saliva-based RT-qPCR by targeting recurrent mutation sites. Rachel E. Ham, Austin R. Smothers, Rui Che, Keegan J. Sell, Congyue Annie Peng, Delphine Dean. doi: https://doi.org/10.1101/2022.03.02.222717851.

Kits that provide for multiplex RT-PCR after reverse transcription, including one-step, [1) One-step RT-PCR Ins214EPE analysis for the detection of the Omicron variant (B.1.1.529) V.1. Nikita Yelynin, Kirill Varchenko, Ksenia Komissarova, Daria Danilenko, Andrey Komissarov, Dmitry Lioznov. https://www.protocols.io/view/one-step-rt-pcr-ins214epe-assay-for-omicron-b-1-1-3by14b2e8vo5/v1; 2) Patent RU 2761025, 07/26/2021, 12/02/2021 3) Patent CN113817868, 07/08/2021, 12/21/2021; 4) Patent CN113215312, 04/28/2021, 08/06/2021; 5) Patent CN113005226, 02/07/2021, 06/22/2021; 6) Patent CN113278733, 05/21/2021, 08/20/2021; 7) IN202221016568, 03/24/2022, 04/08/2022; 8) IN202021026013, 06/19/2020, 06/10/2022] are user-friendly. 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:Ins214EPE 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:Ins214EPE 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:Ins214EPE SARS-CoV-2 mutation with using widely available techniques and available materials.

The technical result is achieved through the use of chemically synthesized oligonucleotides to determine the presence of the S:Ins214EPE SARS-CoV- mutation in a biological sample with a confirmed presence of SARS-CoV-2 RNA, having the following oligonucleotide composition:

Forward primer 214-F - SEQ ID NO: 1,

Reverse primer 214pure-R - SEQ ID NO: 2,

Fluorescent probe 214-Z2 - SEQ ID NO: 3.

The primers are oligonucleotide sequences for amplifying a fragment containing the S:Ins214EPE mutation in biological samples with confirmed SARS-CoV-2 RNA, the 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 based on the known sequence of the gene encoding the SARS-CoV-2 S protein, taken from the NCBI database (https://www.ncbi.nlm.nih.gov/), as a result, a fragment was selected for the detection of the S mutation: Ins214EPE. Primers and a probe for amplification of 149/155 base pairs were selected for the selected fragment: forward primer 214-F - SEQ ID NO:1; reverse primer 214pure-R - SEQ ID NO:2 and fluorescent probe 214-Z2 - 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, fragments of reference genomes from the NCBI database (https://www.ncbi.nlm.nih.gov/) are used, both of the wild-type SARS-CoV-2 virus and of those genovariants, in in which the S:ins214EPE mutation occurs (Omicron (B. 1.1.529)). 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 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:Ins214EPE, 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 forward primer 214-F (SEQ ID NO: 1) and reverse primer 214pure-R (SEQ ID NO: 2) amplify the site in which the S:Ins214EPE 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® 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.

RT-PCR is carried out using the claimed oligonucleotides, primers and probe, presented in Table 1, to detect the S:Ins214EPE mutation of 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 - 0.7 mm each;

- fluorescent probe SEQ ID NO: 3 - 0.2 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) 10 µl cDNA obtained by reverse transcription. 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:Ins214EPE mutation. Samples for which the fluorescence curves cross the threshold line, and, in this case, the kinetics of accumulation of the fluorescent signal is exponential, are positive, that is, they contain the SARS-CoV-2 S:Ins214EPE mutation.

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

Example 1. Obtaining oligonucleotides to determine the S:Ins214EPE mutation of SARS-CoV-2.

To select target sequences - oligonucleotide landing sites, fragments of reference genomes from the NCBI database (https://www.ncbi.nlm.nih.gov/) are used, as wild-type SARS-CoV-2 SARS-CoV-2 from the database, and those genovariants in which the S:ins214EPE mutation occurs (Omicron (B. 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 AlignX, Unipro UGENE, Oligo Calculators (Oligo Calculators) Integrated DNA Technologies, Inc. (Oligo Analyzer (idtdna.com)).Make a list of mutations characteristic of genovariants.Then, oligonucleotide sequences of forward 214-F and reverse 214pure-R primers are selected for significant mutations, as well as the fluorescently labeled probe 214-Z2.

Analysis of these sequences using the Primer BLAST resource showed that the 214-F forward and 214pure-R reverse primers amplify the S:Ins214EPE mutation site with 100% specificity.

Oligonucleotides for detecting the S:Ins214EPE mutation of SARS-CoV-2 forward primer 214-F, reverse primer 214pure-R, fluorescent probe 214-Z2 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:Ins214EPE mutation of SARS-CoV-2 by RT-PCR.

The determination of the S:Ins214EPE mutation of SARS-CoV-2 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 - 0.7 μm each;

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

- 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) 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:Ins214EPE mutation in biological material samples.

Example 3 Detection of SARS-CoV-2 S:Ins214EPE mutation in biological material samples.

To determine the SARS-CoV-2 S:Ins214EPE mutation, 95 samples were selected, with the confirmed presence of SARS-CoV-2 RNA.

For the study, we used clinical material - swabs / discharge of the nasopharynx and oropharynx, with the presence of SARS-CoV-2 RNA confirmed by the AmpliSense® COVID-19-FL reagent kit (Federal Budgetary Institution of the 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 Real-time CFX96 Touch instrument (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 presence of the S:Ins214EPE mutation. For 77 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:Ins214EPE mutation.

For these 77 samples, SARS-CoV-2 S:Ins214EPE 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 18 samples without a fluorescent signal was also confirmed by fragment sequencing.

Thus, 77 samples containing the S:Ins214EPE mutation of the SARS-CoV-2 virus were identified from the entire sample.

Example 4 Detection of SARS-CoV-2 S:Ins214EPE mutation in biological samples.

To determine the SARS-CoV-2 S:Ins214EPE mutation, 5 samples were selected, with the confirmed presence of SARS-CoV-2 RNA.

In this work, samples of clinical material, swabs / discharge of the nasopharynx and oropharynx, with the presence of SARS-CoV-2 RNA confirmed by the AmpliSense® COVID-19-FL reagent kit (Federal Budgetary Institution of the Central Research Institute of Epidemiology of Rospotrebnadzor, Moscow, Russia) were studied. 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 instrument (Qiagen, Germany) 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 presence of the S:Ins214EPE 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:Ins214EPE mutation.

For these 4 samples, SARS-CoV-2 S:Ins214EPE 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, 4 samples containing the S:Ins214EPE SARS-CoV-2 mutation were identified from the entire sample.

Example 5 Detection of SARS-CoV-2 S:Ins214EPE mutation in biological material samples.

To determine the SARS-CoV-2 S:Ins214EPE mutation, 6 samples were selected, with the confirmed presence of SARS-CoV-2 RNA.

In this work, samples of clinical material, swabs / discharge of the nasopharynx and oropharynx, with the presence of SARS-CoV-2 RNA confirmed by the AmpliSense® COVID-19-FL reagent kit (Federal Budgetary Institution of the Central Research Institute of Epidemiology of Rospotrebnadzor, Moscow, Russia) were studied. 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 instrument (Qiagen, Germany) 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 presence of the S:Ins214EPE mutation. The fluorescence curve did not cross the threshold line, indicating that there were no samples containing the S:Ins214EPE SARS-CoV-2 mutation in the sample.

The presence of other SARS-CoV-2 mutations in these 6 samples 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 claimed invention makes it possible to detect the S:Ins214EPE mutation in biological samples with the confirmed presence of SARS-CoV-2 RNA. The synthesized oligonucleotides SEQ ID NO NO: 1-3 do not cross-react with other tested samples, amplify a given site with 100% specificity and allow determining the presence or absence of a significant S:Ins214EPE target in biological material samples.

Claims (3)

1. A set of primers and a probe for determining the mutation S:Ins214EPE SARS-CoV-2, having the following composition: forward primer 214-F - SEQ ID NO: 1; reverse primer 214pure-R - SEQ ID NO: 2; fluorescent probe 214-Z2 - SEQ ID NO: 3. 2. A set of primers and a probe for determining the mutation S:Ins214EPE SARS-CoV-2 according to claim 1, where fragments of the reference SARS-CoV-2 wild-type genomes, Omicron (B.1.1.529) are used to create forward and reverse primers, a fluorescent probe ). 3. A set of primers and a SARS-CoV-2 S:Ins214EPE mutation detection probe 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.