RU2732608C1 - Reagent set for detecting rna of sars-cov-2 virus, causative agent of new coronavirus disease covid-2019, by reverse transcription-polymerase chain reaction in real time - Google Patents

Abstract

FIELD: molecular biology; virology; biotechnology.

SUBSTANCE: invention relates to molecular biology, virology and biotechnology, namely to developing means of detecting and identifying agents of dangerous infectious diseases of viral etiology, and can be used to detect RNA of SARS-CoV-2 virus. Disclosed is a set of oligonucleotide primers and a fluorescent dye-labeled oligonucleotide probe for detecting SARS-CoV-2 virus RNA with the following structure: forward primer SARS-CoV-2_up: 5’-TTGAAGTTTAATCCACCTGCT-3’; reverse primer SARS-CoV-2_low: 5’-ACCGTTCAAGACTCTTTTGC-3’; oligonucleotide probe labeled with fluorescent dye: 5’-(R6G)-CTTATTACAGAGCAAGGGCTGGTGAAG-(RTQ2)-3’. Also disclosed is a reagent kit for detecting SARS-CoV-2 virus RNA by reverse transcription-polymerase chain reaction in real time, containing reverse transcriptase (MMLV-revertase), Tac-polymerase, an equimolar mixture of four deoxynucleotide triphosphates, an internal control sample, a negative control sample, a set of oligonucleotide primers and a fluorescent dye-labeled oligonucleotide probe according to cl.1, positive control sample in form of synthetic RNA, the sequence of which corresponds to hybridisation of oligonucleotide primers, wherein the oligonucleotide primers and the probe are used in a lyophilic dried state.

EFFECT: invention enables to perform reproducible detection of RNA virus SARS-CoV-2 in clinical biological samples with RNA concentration of not less than 1⋅10³ G-E in 1 cm³ without meeting the requirements of "cold chain" during transportation of set.

2 cl, 6 tbl, 4 ex

Description

The invention relates to the field of virology and biotechnology, namely, to the development of means for detecting and identifying causative agents of dangerous infectious diseases of viral etiology, and can be used to detect the RNA of the SARS-CoV-2 virus in clinical biological samples.

Cases of pneumonia of unknown etiology were registered in early December 2019 in Wuhan (PRC), the PRC authorities notified WHO of an outbreak of a new disease on December 31, 2020 [Shih G., Sun L.H. Specter of possible new virus emerging from central China raises alarms across Asia // Washington Post, 8 January 2020 [Electronic resource] URL: https: //flipboard.com@WashPost/specter-of-possible-new-virus-emerging-from -central-china-raises-alarms-across-a / a-LVUQPrl5Qi6rS4m5HImRlg% 3Aa% 3A419161690-66088e4a98% 2Fwashingtonpost.com. (date of access: 09/01/2020); The continuing COVID-2019 epidemic threat of novel coronaviruses in global health - The latest 2019 novel coronavirus outbreak in Wuhan China / D.S. Hu, E.I. Azhar, T.A. Madani et al. // Intern. J. Infect. Dis. - 2020. - Vol. 91. - P. 264-266].

Most of the confirmed cases have been reported from workers and buyers at the South China Seafood Wholesale Market in Wuhan, so an outbreak of the disease, later called COVID-2019 (COrona VIrus Disease), is highly likely to be associated with this seafood market. ... The market was closed only on January 1, 2020, however, as further events showed, this did not prevent the spread of the infection around the world.

According to the WHO decision, the situation with the worldwide spread of COVID-2019 was recognized as a pandemic [WHO declared a coronavirus pandemic [Electronic resource] URL: https // ruslan.rtcom / world / news / 72746 / (date of treatment 03/11/2020)].

Coronaviruses form the subfamily Coronavirinae within the family Coronaviridae, of the order Nidovirales. According to the classification of the International Committee on Taxonomy of Viruses (ICTV), the Coronaviridae family currently includes 4 genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. Coronaviruses are divided into genera based on the principles of phylogeny and determination of the degree of homology for seven highly conserved domains of the replicase polyprotein [De GrootRJ. Family Coronaviridae // King AMQ. Virus taxonomy, the 9th report of the international committee on taxonomy of viruses / ed. by M.J. Adams, E.B. Cartens, E.J. Lefkowitz. - Academic Press: San Diego, CA - 2012. - P. 806-828].

Human disease is caused by representatives of the genera Alphacoronavirus and Betacoronavirus. The genetic diversity of coronaviruses and their variability is provided due to the high frequency of recombination of their genomic RNA and the ability of coronavirus genomes to acquire and lose domains [De Groot R.J. Family Coronaviridae // King AMQ. Virus taxonomy, the 9th report of the international committee on taxonomy of viruses / ed. by M.J. Adams, E.B. Cartens, E.J. Lefkowitz. - Academic Press: San Diego, CA - 2012. - P. 806-828; Nidovirales: evolving the largest RNA virus genome / A.E Gorbalenya, L. Enjuanes, J. Ziebuhr, E.J. Snijder // Virus Res. -2006. - Vol. 117. P. 17-37; Masters P.S. The molecular biology of coronaviruses // Adv. Virus Res. - 2006. - Vol. 66. - P. 193-292; Snijder E.J. Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage // J. Mol. Biol. - 2003. - Vol.331. - P. 991-1004].

It is these factors that contribute to the spontaneous emergence of new coronaviruses that are able to adapt to new hosts and ecological niches, which could potentially be the cause of outbreaks of diseases caused by them.

Whole genome sequencing of the genomic RNA of the new coronavirus was carried out in the first half of January 2020. The genome size was 29903 nucleotide residues (a.i.), the structure of its genome is similar to the structure of the genome of other coronaviruses: 5 '- untranslated region (genome position numbers 1-265 ), OFR-1A (266-13468) and OFR-1B (13468-21555) open reading frames, S gene (21563-25384), OFR-3A gene (25393-26220), E gene (26245-26472), gene M (26523-27191), OFR-6 gene (27202-27387), OFR-7 gene (27394-27759), OFR-8 gene (27894-28259), genES (28274-29533), OFR-10 gene (29556 -29674), untranslated region (29675-29903) - 3 '[CDC Novel coronavirus, (COVID-2019) Wuhan, China [Electronic resource] URL: https://www.cdc.gov/coronavirus/COVID-2019/summary .html. (date of treatment 01/29/2020); Qin A., Hernández J.C. China Reports First Death From New Virus // _ The New York Times, 10 January 2020 [Electronic resource]. URL: htttps: //www.wikizero.com/2019%E2%80%9320_outbreak_of_novel_coronavirus_ (2019-nCoV) (date accessed: 12/01/2020); China reports 136 more cases in two days (in Chinese) // Wuhan Municipal Health Commission [Electronic resource] Archived from the original on January 20, 2020 (date of treatment 01/20/2020)].

The study of the molecular genetic characteristics of the SARS-CoV-2 coronavirus is important in creating effective means of detecting and identifying the causative agent and diagnostics of COVID-2019.

The genomic RNA sequence of SARS-CoV-2 is similar to that of coronaviruses isolated from bats (homology level over 90%) and differs from MERS-CoV and SARS-CoV. The level of homology with SARS-CoV is no more than 80% [CDC Novel coronavirus, (COVID-2019) Wuhan, China [Electronic resource] URL: https://www.cdc.gov/coronavirus/COVID-2019/summary.html. (date of treatment 01/29/2020)].

Analysis of the genome structure suggests that with a high degree of probability the SARS-CoV-2 coronavirus is a product of positive selection due to the mutational variability of the pangolin coronavirus [On the origin and evolution of SARS-CoV-2 / X. Tang, C. Wu, X. Li et al. [Electronic resource] URL: https: //acadimic/oup.com/nsr/advance-article-abstract/doi/10.1093/nsr/nwaa 038/57775463 (date of access 03/09/2020)].

A feature of SARS-CoV-2 is the high affinity of the receptor-binding S-protein for human angiotensin converting enzyme 2 (ACE2), which can be used to enter sensitive cells as well as in the case of infection with the SARS-CoV virus [Uncanny similarity of unique insert in the COVID-2019 spike protein to EQV-1 gpl20 and Gag / P. Pradhan, AK Pandey, A. Mishra et al. // bioRxiv preprint firs posted online on 31.01.2020 [Electronic resource] URL: http://dx.doi.org/10.1101/2020/0130.927871 (date of access 06.02.2020); Snijder E.J. Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage // J. Mol. Biol. - 2003. - Vol. 331. - P. 991-1004].

Genetic analysis of 103 analyzed SARS-CoV-2 virus isolates revealed line L, which is more virulent for humans, and less virulent line S, which is the ancestor of line L. Whole-genome sequencing showed that the genomes of these lines differ by substitutions at positions 8782 (OFR-1A gene ) and 28144 (OFR-8 gene). The substitution at position 8782 (line S-T, line L-C) is synonymous. The substitution at position 28144 (line SC, line LT) leads to the replacement of serine (for line S) at position 84 of the amino acid sequence of the protein encoded by the OFR-8 gene for leucine (for line L) [On the origin and evolution of SARS-CoV- 2 / X. Tang, C. Wu, X. Li et al. [Electronic resource] URL: https: //acadimic/oup.com/nsr/advance-article-abstract/doi/10.1093/nsr/nwaa 038/57775463 (date of access 03/09/2020)].

In the absence of means for the prevention and treatment of the disease caused by SARS-CoV-2, the priority direction of improving the biological protection system of the population of the Russian Federation in the event of a biological emergency as a result of a possible outbreak on its territory is the development of means for identifying and identifying the pathogen. Methods of molecular biology and molecular genetics occupy a leading position in the creation of such means.

The sensitivity of the method used is important. The higher the sensitivity of the research method used, the less the possibility of obtaining false negative results. For COVID-2019, this indicator is especially relevant, since in a number of those infected (especially with the SARS-CoV-2 virus belonging to the S lineage) the disease proceeds without pronounced symptoms, but the pathogen is transmitted.

The aim of the present invention is to develop oligonucleotide primers and create a set of reagents for detecting the RNA of the SARS-CoV-2 virus, the causative agent of the new coronavirus disease COVID-2019, by the method of reverse transcription-real-time polymerase chain reaction (RT-PCR-RT), including the developed oligonucleotide primers as specific components for setting the reaction in the study of clinical and biological samples.

For this, the following studies were carried out:

- the structure of the genome of the SARS-CoV-2 virus was studied, it was compared with the structures of the genomes of the SARS-CoV and MERS-CoV viruses, the variable and conservative regions of the genomes of human pathogenic coronaviruses were identified;

- on the basis of the analysis performed, the choice of the amplified region of the genome was substantiated;

- developed oligonucleotide primers and a probe used as specific components of the reagent kit;

- the composition of specific and nonspecific components of the kit has been substantiated;

- the parameters of the amplification reaction are substantiated;

- the sensitivity, specificity, reproducibility of the RT-PCR-RT method were determined using the specific components of the reagent kit developed by us;

- using the developed set of reagents, the possibility of reproducible detection of SARS-CoV-2 virus RNA in clinical samples obtained from patients with COVID-2019, at a concentration of at least 1⋅10 ³ genome equivalents (GE) in 1 cm ³ , was demonstrated.

The essence of the invention lies in the fact that the detection of the RNA of the SARS-CoV-2 virus, the causative agent of the new coronavirus disease COVID-2019, is carried out by the RT-PCR-RT method using the developed oligonucleotide primers with the activity of forward and reverse primers in the polymerase chain reaction, and oligonucleotide a probe having a specific hybridization region (positions of nucleotide residues 5327-5518) in the genome of the SARS-CoV-2 virus in the region of the OFR-1A gene, which are in a freeze-dried state, and which are specific components of a reagent kit for detection; and allows for reproducible detection of SARS-CoV-2 virus RNA in clinical biological samples with an RNA concentration of at least 1⋅10 ³ H-E in 1 cm ³ .

It should be noted that the use of freeze-dried components (oligonucleotide primers and a probe) allows the reagent kit to be transported without complying with the cold chain requirements.

A set of reagents for detecting RNA of the SARS-CoV-2 coronavirus, the causative agent of COVID-2019, developed at the Vector State Research Center, registered by Roszdravnadzor (11.02.2020, registration No. RZN / 2020/9677) has a similar purpose and can be considered an analogue of the presented inventions [Kleshchenko E., Torgashev A. Test for coronavirus for Russia [Electronic resource] URL: https: //pcr.neus//state/test-na-koronavirus-dlya-rossii (date of access 03/17/2020)].

The features of the presented invention, which differ from the analogue, include:

- completeness of the set. The set we declare contains all the specific and non-specific components required for the study of samples. The analogue kit contains no reagents for the extraction of viral RNA and reverse transcription, which creates inconvenience in the process of laboratory diagnostics;

- in the claimed set of reagents, synthetic RNA is used as a positive control sample (PCO), the nucleotide sequence of which corresponds to a fragment of the OFR-1A gene sequence of the genome of the SARS-CoV-2 virus, which allows you to control the stage of reverse transcription. A recombinant plasmid with an insert of cDNA of genomic RNA is used as a PCO in the analogue kit, and the analogue kit does not contain: a reagent allowing to control the stage of reverse transcription and an internal control sample (ICS);

- the oligonucleotide primers and the probe of the claimed set are in a lyophilized state, while in the analogue set - in a liquid state, which significantly complicates the conditions of storage and transportation;

- the sensitivity of the claimed set is 1⋅10 ³ G-E⋅cm ^-3 . According to the passport data, the sensitivity of the RT-PCR-RT method of the analogue kit is 1⋅10 G-E⋅cm ^-3 . The lower sensitivity of the analogue kit can lead to underdiagnosis of the disease due to false negative results.

The structure of oligonucleotide primers and probe specific to the specified region of the genome of the SARS-CoV-2 virus presented in this application has no analogues in the world.

The technical result of the invention is the reproducible detection of SARS-CoV-2 coronavirus RNA in clinical and biological samples, at a concentration of at least 1⋅10 ³ G-E⋅cm- ³ without observing the cold chain requirements when transporting the kit.

The specified technical result of the invention is achieved due to:

- selection of oligonucleotide primers with specific hybridization regions in the OFR-1A open reading frame of the SARS-CoV-2 coronavirus, synthesis of oligonucleotide primers with the activity of forward and reverse primers in PCR;

- completeness of the kit, including specific and non-specific components required for the study of samples;

- lyophilic drying of the synthesized oligonucleotide primers and probe.

The selection of oligonucleotide primers was carried out using the Vector NTI software. The developed primers are species-specific for the SARS-CoV-2 coronavirus.

The primers were synthesized by solid-phase chemical synthesis followed by purification by polyacrylamide gel electrophoresis and high-performance liquid chromatography.

To increase the preservation of these specific components of the reagent kit and to ensure the possibility of transporting the reagent kit without observing the requirements of the "cold chain", the synthesized oligonucleotide primers and probe were lyophilized, which allows the guaranteed storage of the reagent kit at + 4 ° C for 12 months.

The composition of the proposed set of reagents is presented in table 1.

The proposed technical solution is new, since a set of reagents is unknown in publicly available sources in the Russian Federation, which includes all the necessary specific and nonspecific components that provide reproducible detection of SARS-CoV-2 virus RNA by RT-PCR-RT, with its concentration in the samples being at least 1⋅10 ³ G-E⋅cm ^-3 without observing the requirements of the "cold chain" when transporting the kit.

The proposed technical solution has an inventive step, since the proposed oligonucleotide primers and a probe for detecting SARS-CoV-2 virus RNA by RT-PCR-RT were developed as a result of the selection and synthesis of primers having the OFR-1A gene as a hybridization region in the virus genome SARS-CoV-2, which ensures the detection of RNA of the specified virus by RT-PCR-RT with obtaining reproducible results in preparations with an RNA concentration of at least 1.0⋅10 ³ G-E⋅cm ^-3 .

The proposed technical solution is industrially applicable, since typical biotechnological equipment, as well as reagents and materials widely used in the biotechnological industry, can be used for its implementation.

The claimed set of reagents, based on the developed primers and the oligonucleotide probe, is advisable to use in the study of material from patients, as well as in case of suspicions of COVID-2019 and indication of the SARS-CoV-2 virus in environmental samples, for epidemiological monitoring.

The possibility of implementing the claimed invention is shown by the following examples.

Example 1. Evaluation of the specificity of the developed oligonucleotides in relation to closely related heterologous coronaviruses

To assess the specificity of the developed oligonucleotides for closely related heterologous coronaviruses, using the AlignX program of the VectorNTIv.11 package, multiple alignments of the genome of the coronavirus and heterologous coronaviruses were prepared. The results of the theoretical analysis of specificity are presented in Table 2.

The results presented in Table 2 allow us to conclude that, due to the significant number of nucleotide substitutions, amplification of heterologous coronaviruses using the developed primers and probe is impossible.

Example 2. Experimental study of the analytical sensitivity and reproducibility of the RT-PCR-RT method using the developed set of reagents

The sensitivity, specificity, reproducibility of the RT-PCR-RT method when using the specific components of the reagent kit developed by us were determined using an artificially created GIK, which is a synthetic RNA characterized by the following primary structure:

Determination of analytical sensitivity by biological (virological) method was carried out as follows. Samples containing different amounts of GE were prepared by dilutions performed in two steps. The resulting preparations were analyzed using RT-PCR-RT using our developed oligonucleotide primers and probe. The analysis results are presented in Table 3.

The calculation results presented in Table 3 allow us to conclude that the use of the oligonucleotide primers and the RT-PCR-RT probe we have developed will make it possible to specifically detect SARS-CoV-2 virus RNA in RNA preparations with a concentration of at least 1.0⋅10 ³ G -E⋅cm ^-3 . The total number of positive determinations for a concentration of 1.0⋅10 ³ G-E⋅cm ^-3 is 20 out of 20, which corresponds to a reproducibility of at least 95% [Genes BC Some simple methods of cybernetic processing of diagnostic and physiological research data. - M .: Nauka, 1967. - 208 p.].

Thus, the analytical sensitivity value is 1.0⋅10 ³ G-E⋅cm ^-3 with a reproducibility of at least 95%.

Example 3. Experimental study of the specificity of RT-PCR-RT using the developed set of reagents

To experimentally study the specificity of RT-PCR-RT using the developed set of reagents, we evaluated nucleic acid preparations obtained from viruses representing different taxonomic groups of causative agents of acute respiratory diseases: coronaviruses SARS-CoV, MERS-CoV, CoV-OC43, CoV-229E, viruses influenza A (antigenic subtypes H1N1, H2N3, H5N1). Analytes provided by the Institute. N.F. Gamalei.

The criterion of specificity was the presence of a fluorescent signal in the R6G channel in the analysis of RNA preparations of the SARS-CoV-2 virus and the absence of a signal in this channel in the analysis of heterologous RNA and DNA.

The analysis was performed in triplicate using the "single difference" criterion - analyte detected / analyte not detected.

The result was considered truly positive if the analyte was detected in all tubes with the SARS-CoV-2 GIK of the MERS virus via the ROX channel.

The result was considered false negative if the analyte was not detected in any of the tubes with the SARS-CoV-2 GIK through the ROX channel.

The result was considered truly negative in the absence of detection of the analyte in all tubes with RNA of the remaining targets through the R6G and ROX channels.

The result was considered false positive if the analyte was detected in any of the tubes with RNA of the remaining targets through the R6G or ROX channels.

The criterion according to which the reagent kit was considered to pass the test for analytical specificity was considered a truly positive result for SARS-CoV-2 virus RNA with a truly negative result for all other analytes.

The experimental results presented in Table 4 confirm the specificity of the tested reagent kit for SARS-CoV-2 virus. Strains of heterologous microorganisms are not detected.

Example 4. Analysis of clinical samples obtained from patients with COVID-2019 and persons with suspected COVID-2019 using RT-PCR-RT using the developed set of reagents

In the experiment, the presence of SARS-CoV-2 virus RNA was determined in clinical samples from patients with COVID-2019 and persons with suspected COVID-2019 (blood serum and nasopharyngeal lavages). The research results are presented in table 5.

Thus, the set of reagents developed at the 48 Central Research Institute of the Ministry of Defense of the Russian Federation for diagnosing the disease COVID-2019 allows for reproducible detection of SARS-CoV-2 virus RNA when its concentration in the test samples is at least 1⋅10 ³ genome equivalents in 1 cm ³ without observing the requirements of the "cold chain" when transporting the kit.

Claims (8)

1. A set of oligonucleotide primers and an oligonucleotide probe labeled with a fluorescent dye for detecting the RNA of the SARS-CoV-2 virus with the following structure: - forward primer SARS-CoV-2_up: 5'-TTGAAGTTTAATCCACCTGCT-3 '; - reverse primer, SARS-CoV-2_low: 5'-ACCGTTCAAGACTCTTTTGC-3 '; - labeled with a fluorescent dye oligonucleotide probe: 5 '- (R6G) -CTTATTACAGAGCAAGGGCTGGTGAAG- (RTQ2) -3'. 2. A set of reagents for the detection of SARS-CoV-2 virus RNA by the method of reverse transcription-polymerase chain reaction in real time, containing reverse transcriptase (MMLV-revertase), Tac-polymerase, an equimolar mixture of four deoxynucleotide triphosphates, an internal control sample, a negative control sample, a set of oligonucleotide primers and an oligonucleotide probe labeled with a fluorescent dye according to claim 1, a positive control sample, which is a synthetic RNA, the sequence of which corresponds to the hybridization region of the oligonucleotide primers, while the oligonucleotide primers and the probe are used in a freeze-dried state.