RU2764021C1 - METHOD FOR DETECTING SARS-CoV-2 VIRUS RNA IN ULTRA-LOW CONCENTRATIONS AND SPECIFIC OLIGONUCLEOTIDES FOR USE IN THE METHOD - Google Patents

Abstract

FIELD: genetic engineering; biotechnology.

SUBSTANCE: group of inventions relates to the field of genetic engineering and biotechnology. A method for detecting SARS-CoV-2 virus RNA, a specific oligonucleotide and a kit for use in the method for detecting SARS-CoV-2 virus RNA are proposed. The SARS-CoV-2 virus RNA detection method uses guide RNAs and specific oligonucleotides for pre-amplification.

EFFECT: inventions make it possible to effectively detect the RNA of the SARS-CoV-2 virus after specific amplification of fragments of the genome of this virus and also provide detection of single copies of the SARS-CoV-2 virus RNA.

5 cl, 11 dwg, 5 tbl, 5 ex

Description

Technical field

The invention relates to the field of genetic engineering and biotechnology, namely to the number of guide RNA tools that can be used in CRISPR-Cas12 systems as part of ribonucleoprotein complexes for the detection (detection, detection) of SARS-CoV-2 virus RNA, as well as to diagnostic methods and sets.

EFFECT: invention allows to detect single copies of SARS-CoV-2 virus RNA in vitro.

The guide RNAs described in this application can be used to detect SARS-CoV-2 virus RNA after specific amplification of fragments of the SARS-CoV-2 genome. Amplification in this case can be carried out in various ways, including polymerase chain reaction (PCR); loop isothermal amplification (LAMP); helicase-dependent amplification (HDA); recombinase-mediated amplification (RPA); strand displacement amplification (SDA); nucleic acid sequence based amplification (NASBA); transcription-mediated amplification (TMA); nicking enzyme mediated amplification (NEAR); circular amplification (RCA) and many other types of amplification.

The guide RNAs described in this application can be used to develop highly sensitive and high-tech next-generation diagnostic systems based on CRISPR technologies to improve methods for diagnosing infectious diseases, including the development of methods for diagnosing newly emerging infections.

State of the art

To solve the epidemiological problems of deciphering outbreaks of infectious diseases, identifying and identifying the pathogen, as well as detecting specific bacterial and viral genes, it is necessary to develop and implement modern technologies of molecular epidemiology into the practice of surveillance and monitoring services. One such technology is the use of genetic editing elements of the CRISPR/CAS system. This technology is developing quite effectively in relation to the creation of drugs for the treatment of certain diseases, despite a number of difficulties associated with the occurrence of unforeseen mutations. With in-depth studies in the field of application of the CRISPR / CAS system, it was found that it can be used for subtle diagnostic procedures in identifying the pathogen / her infection in humans, as well as their genotyping.

In 2018, it was shown that one of the enzymes of the CRISPR system, Cas12, after recognizing its target DNA target, begins to nonspecifically hydrolyze single-stranded DNA. This property of Cas12 can be used as an indicator of the presence of a specific target, such as the genome of a virus or bacteria. The researchers used this discovery to create a nucleic acid detection technology platform known as DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter). The proposed platform combines the Cas12a nuclease, its nucleic acid-specific guide RNA, and a fluorescent reporter molecule. For the first time, DETECTR technology was used to detect and genotype the human papillomavirus. DETECTR allowed differentiation of HPV16 and HPV18 in crude DNA extracts from cultured human cells and clinical specimens within 1 h. At the same time, DETECTR correctly (comparable to the results of PCR analysis) identified HPV16 in 25 and HPV18 in 23 out of 25 clinical samples (Chen JS, Ma E., Harrington LB, et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity Science 2018 360(6387): 436-9 doi: 10.1126/science.aar6245).

An equally important application of the CRISPR/Cas system is the identification of pathogens and the detection of specific bacterial genes using the SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform. The proposed platform combines the Cas13a nuclease, its nucleic acid-specific guide RNA, and a fluorescent reporter molecule. The Cas13a complex binds and cleaves the pre-amplified target nucleic acid with high specificity. Using SHERLOCK, it was possible to correctly differentiate closely related Zika and Dengue virus strains (Gootenberg JS, Abudayyeh OO, Lee JW, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017; 356(6336): 438-42. doi: 10.1126/science.aam9321).

SHERLOCK has been improved (SHERLOCKv2) and allowed to detect up to 4 targets in one analysis. Multiplexing was achieved by combining several Cas13 nucleases and a Cas12 nuclease with specific fluorescent reporter complexes that allowed signal detection at different wavelengths. Quantitative detection was achieved by optimizing the concentrations of oligonucleotides used in the pre-amplification step so that input signal and signal intensity are closely correlated over a wide range of sample concentrations. Increased sensitivity was achieved by adding Csm6 to increase the intensity of fluorescent reporter cleavage. It should be noted that SHERLOCKv2 is a portable analysis due to the fact that the detection of fluorescence readings in the proposed technology is replaced by visual detection on test strips (Gootenberg JS, Abudayyeh O.O., Kellner MJ, et al. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6 Science 2018 360(6387): 439-44 doi: 10.1126/science.aaq0179).

Based on SHERLOCK, the HUDSON (heating unextracted diagnostic samples to obliterate nucleases) technology was developed, which eliminates the need for extraction of nucleic acids and allows the detection of pathogens directly in patient biological samples (blood, serum or plasma sample, blood cells, saliva, sputum, lymphoid tissues, tissues of hematopoietic organs and other biological materials). At HUDSON, heat and chemical reduction inactivate nucleases present in high concentrations in patient biological samples, after which the viral particles are lysed, releasing nucleic acids into solution. HUDSON allows detection of Dengue virus within 2 hours with high sensitivity in samples of whole blood, serum and saliva of patients. In addition, HUDSON allows you to differentiate four Dengue virus serotypes and identify the 6 most common HIV reverse transcriptase mutations (Myhrvold C., Freije C.A., Gootenberg JS, et al. Field-deployable viral diagnostics using CRISPR-Casl3. Science. 2018; 360(6387): 444-8 doi: 10.1126/science.aas8836).

In this regard, the task of developing new effective methods for detecting nucleic acids of pathogens of infectious diseases based on genetic technologies, such as CRISPR/Cas, is extremely urgent.

During the study of the prior art, scientific articles were found describing the development of technologies for the detection of SARS-CoV-2 virus RNA in vitro, based on the use of proteins of directed genome editing systems.

J.P. Broughton and co-authors described the DETECTR technology for detecting SARS-CoV-2 virus RNA, where the N and E genes of SARS-CoV-2 served as a target for amplification by the method of isothermal amplification combined with reverse transcription (RT-LAMP), and the detection took place with Cas12a in test strip format. The sensitivity of the described method varied from 70 to 300 copies of SARS-CoV-2 virus RNA per reaction [J.P. Broughton, X. Deng, G. Yu, C.L. Fasching, V. Servellita, J. Singh, et al., CRISPR Casl2-based detection of SARS-CoV-2, Nature Biotechnology 38 (2020) 870-874, https://doi.org/10.1038/s41587-020- 0513-4].

Cas12a has also been used to detect SARS-CoV-2 virus RNA in a method called AIOD-CRISPR. The N gene was chosen as a target, the amplification of a fragment of which was carried out in the course of an isothermal reaction combined with reverse transcription (RT-RPA). The sensitivity of AIOD-CRISPR was 10-100 copies of SARS-CoV-2 virus RNA per reaction [X. Ding, K. Yin, Z. Li, R. Lalla, E. Ballesteros, M Sfeir, C. Liu, Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Casl2a assay. Nat. commun. 2020 (18) (2020) https://doi.org/10.1038/s41467-020-18575-6].

In addition, the ENHANCE technology was developed, which makes it possible to detect from 3 to 300 copies of SARS-CoV-2 virus RNA per reaction, in which the preliminary amplification of the N gene fragment was carried out by the method of isothermal amplification combined with reverse transcription (RT-LAMP), followed by detection amplification products with modified Cas12a [LT Nguyen, V.M. Smith, R.K. Jain, Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection, Nat. commun. 11 (4906) (2020), https://doi.org/10.1038/s41467-020-18615-l].

L.U. Guo and co-authors in their work described the CASdetec technology, which made it possible to detect 100-500 copies of SARS-CoV-2 virus RNA per reaction. The technology was based on isothermal amplification using recombinase coupled with reverse transcription (RT-RAA). The SARS-CoV-2 RdRp gene fragment served as the target for amplification. Amplification products were detected using Cas12b [L.u. Guo, X. Sun, X. Wang, C. Liang, H. Jiang, Q. Gao, et al., SARS-CoV-2 detection with CRISPR diagnostics, Cell Discov 6 (1) (2020), https:// doi.org/10.1038/s41421-020-0174-y].

When using the SHERLOCK platform to detect the RNA of the SARS-CoV-2 virus, the S and Orflab genes were described as a target. Preamplification was performed using isothermal amplification combined with reverse transcription (RT-RPA), before detection with Cas13a, an additional step of in vitro transcription was performed. The SHERLOCK technology made it possible to detect 10-100 copies of SARS-CoV-2 virus RNA per reaction [F. Zhang, O.O. Abudayyeh, J.S. Gootenberg, C. Sciences, L. Mathers, A protocol for detection of COVID-19 using CRISPR diagnostics, Bioarchive. (2020) 1 8].

In addition to the methods listed above, the FELUDA diagnostic platform was proposed for the detection of SARS-CoV-2 virus RNA. Detection of ARS-CoV-2 virus S RNA (NSP8 and N genes) occurred after reverse transcription with Cas9. The sensitivity of the method was estimated at 3.16-10 copies of SARS-CoV-2 virus RNA per reaction [S.M. Mohd. Azhar, Rhythm Phutela, Asgar Hussain Ansari, Dipanjali Sinha, Namrata Sharma, Manoj Kumar, Meghali Aich, Saumya Sharma, Khushboo Singhal, Harsha Lad, Pradeep K. Patra, Govind Makharia, Giriraj Ratan Chandak, Debojyoti Chakraborty, Rapid, field-deployable nucleobase detection and identification using FnCas9, BioRxiv. (2020). doi:: https://doi.org/10.1101/2020.04.07.028167].

Based on this, a technical problem arises, which consists in the need to obtain guide RNAs to detect single copies (less than 3 copies of RNA per reaction) of SARS-CoV-2 virus RNA in vitro using Casl2 and to develop appropriate methods for detecting SARS-CoV-2 virus RNA .

Essence disclosure

The proposed technology is promising for a variety of applications, including DNA/RNA quantification, fast multiplex expression detection, and other types of sensitive detection, such as detection of sample contamination with nucleic acids. CRISPR/Cas based technology is a feature rich, error tolerant DNA detection technology suitable for rapid diagnosis including infectious diseases, genotyping of infectious agents, and detection of antibiotic resistance genes in bacterial pathogens.

The application of the proposed technology makes it possible to create a new generation of diagnostic systems that will have the following properties:

высокая чувствительность;

high sensitivity;

возможность проведения диагностики у постели больного;

the possibility of carrying out diagnostics at the patient's bedside;

возможность проведения диагностики в полевых условиях без применения специализированного высокотехнологичного оборудования;

the possibility of carrying out diagnostics in the field without the use of specialized high-tech equipment;

скорость и простота анализа;

speed and simplicity of analysis;

сниженная стоимость анализа;

reduced cost of analysis;

отсутствие необходимости оснащения диагностической лаборатории дорогостоящим оборудованием;

no need to equip the diagnostic laboratory with expensive equipment;

отсутствие необходимости проведения выделения нуклеиновых кислот возбудителя.

no need to isolate the nucleic acids of the pathogen.

The invention relates to novel guide RNA tools that can be used in CRISPR-Cas12 systems for ultrasensitive detection, identification, detection or detection of SARS-CoV-2 virus RNA in biological samples.

The technical objective of the proposed invention is the development of new tools - guide RNAs that can be used in CRISPR-Cas12 systems with Casl2 proteins, for example, LbCpf1 from Lachnospiraceae, for ultrasensitive detection of SARS-CoV-2 virus RNA.

When implementing the present invention, according to the set of essential features given in the claims, an unexpected technical result is achieved - the possibility of ultrasensitive detection of SARS-CoV-2 virus RNA up to single (1.64) copies of RNA in one reaction. EFFECT: invention provides up to 10 times higher efficiency of SARS-CoV-2 virus RNA detection.

The technical result is achieved due to:

разработки молекул направляющих РНК, которые могут быть использованы в системах CRISPR-Cas12 для ультра чувствительного выявления РНК вируса SARS-CoV-2, где указанные направляющие РНК выбраны из последовательностей SEQ ID NO: 1-5, способны связываться с целевыми высоко консервативными участками РНК вируса SARS-CoV-2, содержат РНК-шпильку, которая распознается РНК-направляемой ДНК-эндонуклеазой LbCpf1 из Lachnospiraceae, с обеспечением выявления единичных копий РНК вируса SARS-CoV-2.

development of guide RNA molecules that can be used in CRISPR-Cas12 systems for ultra-sensitive detection of SARS-CoV-2 virus RNA, where these guide RNAs are selected from the sequences of SEQ ID NO: 1-5, are able to bind to highly conserved target regions of the virus RNA SARS-CoV-2 contain a hairpin RNA that is recognized by the RNA-directed DNA endonuclease LbCpf1 from Lachnospiraceae, allowing detection of single copies of the SARS-CoV-2 virus RNA.

применения РНК-направляемой ДНК-эндонуклеазы LbCpf1 из Lachnospiraceae, полученной согласно способу, разработанному авторами ранее (Патент RU №2707542, дата приоритета 28.03.2019), для создания рибонуклеопротеиновых комплексов (РПК) системы CRISPR/Cas, пригодных для детекции РНК вируса SARS-CoV-2 в ультра низких концентрациях (единичные копии).

application of RNA-directed DNA endonuclease LbCpf1 from Lachnospiraceae, obtained according to the method developed by the authors earlier (Patent RU No. 2707542, priority date 03/28/2019), to create ribonucleoprotein complexes (RPK) of the CRISPR/Cas system suitable for detecting SARS-virus RNA CoV-2 at ultra low concentrations (single copies).

разработки набора специфических олигонуклеотидов, выбранных из SEQ ID NO: 6-9, для предварительной амплификации фрагментов генома вируса SARS-CoV-2.

development of a set of specific oligonucleotides selected from SEQ ID NO: 6-9 for preliminary amplification of SARS-CoV-2 virus genome fragments.

оптимизации условий проведения предварительной амплификации фрагментов генома вируса SARS-CoV-2.

optimization of conditions for preliminary amplification of SARS-CoV-2 virus genome fragments.

определения условий проведения ультрачувствительной детекции РНК вируса SARS-CoV-2 и установления последовательности стадий метода.

determining the conditions for ultrasensitive detection of SARS-CoV-2 virus RNA and establishing the sequence of the method steps.

The guide RNAs of the present invention correspond to highly conserved fragments of the SARS-CoV-2 virus genome. Most preferred are guide RNAs recognized by the RNA-guided DNA endonuclease LbCpf1 from Lachnospiraceae, characterized by having or containing a nucleotide sequence selected from:

SEQ ID NO: 1;

SEQ ID NO: 1;

SEQ ID NO: 2;

SEQ ID NO: 2;

SEQ ID NO: 3;

SEQ ID NO: 3;

SEQ ID NO: 4;

SEQ ID NO: 4;

SEQ ID NO: 5;

SEQ ID NO: 5;

или идентичной любой из них по меньшей мере на 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% или 99%,

or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% identical to any of them , 94%, 95%, 96%, 97%, 98% or 99%,

или комплементарной любой из них,

or complementary to any of them,

или гибридизующейся с любой из них в строгих условиях. Набор специфических олигонуклеотидов для проведения предварительной амплификации фрагмента генома вируса SARS-CoV-2, согласно настоящему изобретению, соответствуют высоко консервативным участкам генома вируса SARS-CoV-2. Наиболее предпочтительны олигонуклеотиды, характеризующиеся, имеющие или содержащие нуклеотидную последовательность, выбранную из:

or hybridizing with any of them under stringent conditions. The set of specific oligonucleotides for pre-amplification of a fragment of the genome of the SARS-CoV-2 virus, according to the present invention, correspond to highly conserved regions of the genome of the SARS-CoV-2 virus. Most preferred are oligonucleotides characterized by having or containing a nucleotide sequence selected from:

SEQ ID NO: 6;

SEQ ID NO: 6;

SEQ ID NO: 7;

SEQ ID NO: 7;

SEQ ID NO: 8;

SEQ ID NO: 8;

SEQ ID NO: 9;

SEQ ID NO: 9;

или идентичной любой из них по меньшей мере на 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% или 99%,

or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% identical to any of them , 94%, 95%, 96%, 97%, 98% or 99%,

или комплементарной любой из них, или гибридизующейся с любой из них в строгих условиях.

or complementary to any of them, or hybridizing with any of them under stringent conditions.

According to the proposed invention, ribonucleoprotein complexes (RPK) are obtained, consisting of at least one guide RNA and one RNA-directed DNA endonuclease of the CRISPR/Cas system (LbCpf1 from Lachnospiraceae), suitable for use to detect SARS-CoV-2 virus RNA in ultra-low concentrations (single copies).

PRP preparations are solutions containing a guide RNA selected from SEQ ID NO: 1-5 combined with a CRISPR/Cas system protein (LbCpf1 from Lachnospiraceae) or freeze-dried PRP.

The resulting guide RNAs can be used as part of a SARS-CoV-2 virus RNA detection kit, with instructions for use.

SARS-CoV-2 virus RNA detection method:

i) performing pre-amplification of the sample material from a patient suspected of containing the SARS-CoV-2 virus using one or more specific oligonucleotides selected from SEQ ID NO: 6-9, in order to obtain a target of a pre-amplified fragment of the genome of the SARS-CoV-2 virus ,

ii) preparing a detection reaction mixture containing the target obtained in step (i); a ribonucleoprotein complex of the CRISPR/Cas system formed from the RNA-guided DNA endonuclease LbCpf1 from Lachnospiraceae and at least one guide RNA with SEQ ID NOs: 1-5; fluorescent probe and detection buffer,

iii) carrying out 30-60 detection cycles in the reaction mixture obtained in step (ii) in a cycler, thus detecting SARS-CoV-2 virus RNA.

Pre-amplified fragments of the genome of the SARS-CoV-2 virus, according to the proposed method, can be represented by fragments of 260 bp in size. genome of the SARS-CoV-2 virus.

The proposed method ensures the detection of SARS-CoV-2 virus RNA at ultra-low concentrations, down to single copies.

The specific oligonucleotide for use in the method is selected from the group SEQ ID NO: 6-9 and is used for pre-amplification of highly conserved fragments of the SARS-CoV-2 virus genome that are recognized by ribonucleoprotein complexes.

The kit for use in the method for detecting RNA of the SARS-CoV-2 virus contains a ribonucleoprotein complex of the CRISPR/Cas system formed from the RNA-directed DNA endonuclease LbCpf1 from Lachnospiraceae, and at least one guide RNA with SEQ ID NO: 1-5; fluorescent probe, detection buffer and instructions for use.

The kit may additionally include components for pre-amplification of highly conserved fragments of the SARS-CoV-2 virus genome, including one or more specific oligonucleotides selected from SEQ ID NOs: 6-9.

At the same time, at least one guide RNA in the kit can be complexed with a protein of the CRISPR/Cas system (LbCpf1 from Lachnospiraceae) in one container or separately in different containers.

The proposed technology makes it possible to determine single copies of SARS-CoV-2 virus RNA in biological samples of a patient selected from fluid and/or tissue suspected to contain the SARS-CoV-2 virus. The biological sample can be a sample of blood, blood serum or plasma, cerebrospinal fluid, blood cells, saliva, sputum, lymphoid tissues, tissues of hematopoietic organs and other biological materials from a patient that can be used to analyze for the presence of SARS-CoV-2 virus RNA .

Brief description of the drawings

Fig. 1. Visualization of the amplified fragment of the genome of the SARS-CoV-2 virus (260 bp in size) after preliminary amplification by agarose gel electrophoresis, where the numbers 1-8 indicate:

1 - The product obtained during the amplification of 2000 fg of the pGEM-T-SARS-CoV-2 model matrix;

2 - The product obtained during the amplification of 200 fg of the pGEM-T-SARS-CoV-2 model matrix;

3 - The product obtained during the amplification of 20 fg of the pGEM-T-SARS-CoV-2 model matrix;

4 - The product obtained during the amplification of 2 fg of the pGEM-T-SARS-CoV-2 model matrix;

5 - The product obtained during the amplification of 0.2 fg of the pGEM-T-SARS-CoV-2 model matrix;

6 - The product obtained during the amplification of 0.02 fg of the pGEM-T-SARS-CoV-2 model matrix;

7 - The product obtained during the amplification of 0.002 fg of the pGEM-T-SARS-CoV-2 model matrix;

8 - The product obtained during the amplification of 0.0002 fg of the pGEM-T-SARS-CoV-2 model matrix;

9 - Negative control, not containing the pGEM-T-SARS-CoV-2 model matrix;

M - molecular weight standards: from bottom to top 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 2000, 3000 bp (GeneRuler 100 bp Plus, Thermo Fisher Scientific, USA).

Fig. 2. Real-time fluorescence profile of a pre-amplified SARS-CoV-2 target treated with a ribonucleoportein complex containing SARS-CoV-2 crRNA #26333 guide RNA and LbCpf1 protein.

Fig. 3. Real-time fluorescence profile of a pre-amplified SARS-CoV-2 target treated with a ribonucleoportein complex containing SARS-CoV-2 crRNA #26413 guide RNA and LbCpf1 protein.

Fig.4. Real-time fluorescence profile of a pre-amplified SARS-CoV-2 target treated with a ribonucleoportein complex containing SARS-CoV-2 crRNA #26432 guide RNA and LbCpf1 protein.

Fig. 5. Real-time fluorescence profile of a pre-amplified SARS-CoV-2 target treated with a ribonucleoportein complex containing SARS-CoV-2 crRNA #26451 guide RNA and LbCpf1 protein.

Fig. 6. Real-time fluorescence profile of a pre-amplified SARS-CoV-2 target treated with a ribonucleoportein complex containing SARS-CoV-2 crRNA #26460 guide RNA and LbCpf1 protein.

Fig. Figure 7. Fluorescence values at the end point (60 assay cycle, 60 minutes) for a pre-amplified SARS-CoV-2 target treated with ribonucleoportein complexes containing the LbCpf1 protein and guide RNA SARS-CoV-2 crRNA #26333, SARS-CoV-2 crRNA No. 26413, SARS-CoV-2 crRNA No. 26432, SARS-CoV-2 crRNA No. 26451, SARS-CoV-2 crRNA No. 26460, respectively.

Fig. Fig. 8. Visualization of amplification products obtained during the amplification of SARS-CoV-2 fragments using 10 clinical samples, where M is the molecular weight standards: from bottom to top 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 , 1200, 1500, 2000, 3000 bp (GeneRuler 100 bp Plus, Thermo Fisher Scientific, USA).

Fig. 9. Endpoint fluorescence values (60 assay cycle, 60 minutes) for a pre-amplified SARS-CoV-2 target (10 independent clinical specimens) treated with ribonucleoportein complexes containing the LbCpf1 protein and SARS-CoV-2 crRNA guide RNA #26333, SARS-CoV-2 crRNA #26413, SARS-CoV-2 crRNA #26432, SARS-CoV-2 crRNA #26451, SARS-CoV-2 crRNA #26460, respectively.

Fig. Fig. 10. Visualization of the amplification products obtained during the amplification of SARS-CoV-2 fragments using tenfold dilutions of the RNA preparation isolated from the clinical sample of the patient, where M molecular weight standards: from bottom to top 100, 200, 300, 400, 500, 600, 700 , 800, 900, 1000, 1200, 1500, 2000, 3000 bp (GeneRuler 100 bp Plus, Thermo Fisher Scientific, USA).

Fig. 11. Fluorescence values at the endpoint (60 assay cycle, 60 minutes) for a pre-amplified SARS-CoV-2 target (ten-fold dilutions of an RNA preparation isolated from a clinical patient specimen) treated with ribonucleoportein complexes containing the LbCpf1 protein and SARS- crRNA guide RNAs. CoV-2 #26333, SARS-CoV-2 crRNA #26413, SARS-CoV-2 crRNA #26432, SARS-CoV-2 crRNA #26451, SARS-CoV-2 crRNA #26460, respectively.

EXAMPLES OF CARRYING OUT THE INVENTION

EXAMPLE 1: SELECTION OF TARGET SEQUENCES IN THE SARS-COV-2 VIRUS GENOME TO GENERATE GUIDE RNA

For the selection of target sequences in the genome of the SARS-CoV-2 virus, modern algorithms for in silico analysis of nucleotide sequences and programs that are in the public domain, including Benchling (https://www.benchling.com/molecular-biology) were used to create guide RNAs. /). A list of regions in the genome of the SARS-CoV-2 virus was compiled with a theoretically calculated probability of their cleavage in highly conserved regions (Table 1. List of regions of the SARS-CoV-2 virus genome with a theoretically calculated probability of their cleavage by LbCpf1 from Lachnospiraceae).

Guide RNAs specifically recognizing fragments in the genome of the SARS-CoV-2 virus are represented by unique sequences of SEQ ID NOs: 1-5.

EXAMPLE 2: PREPARATION OF MATERIAL FOR SARS-COV-2 VIRUS RNA DETECTION BY THE PRE-AMPLIFICATION METHOD

Preparation of material for the detection of SARS-CoV-2 virus RNA is carried out by the method of preliminary amplification combined with reverse transcription (RT-PCR). Synthetic RNA obtained by in vitro transcription is used as a model template for a fragment of the genome of the SARS-CoV-2 virus of a biological sample. As a template for the synthesis of model RNA, plasmid DNA pGEM-T-SARS-CoV-2 is used, which contains a fragment of the gene encoding the E protein of the SARS-CoV-2 virus (260 bp in size).

Synthesis of the cDNA fragment of the SARS-CoV-2 virus genome is carried out by reverse transcription using the specific oligonucleotide E-pr_Revl with SEQ ID NO: 7 (Table 2. Specific oligonucleotides for preliminary amplification of SARS-CoV-2 virus genome fragments) and Superscript™ reverse transcriptase II (Thermo Fisher Scientific, USA) at 42°С.

Pre-amplification of a fragment of the genome of the SARS-CoV-2 virus is carried out using specific oligonucleotides E-pr_Forl and E-pr_Revl with SEQ ID NO: 6 and SEQ ID NO: 7, respectively, shown in Table 2. Specific oligonucleotides for pre-amplification of fragments of the SARS virus genome -CoV-2. Amplification products encoding a fragment of the ARS-CoV-2 virus S genome are obtained in an amplification reaction using PCR mixture-2 blue (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) and specific oligonucleotides (GenTerra, Russia) listed in Table 2. Specific oligonucleotides for preliminary amplification of SARS-CoV-2 virus genome fragments. The size of the amplified fragments is 260 base pairs. Temperature profile of amplification:

1. denaturation: 95°C for 3 minutes;

2. 35 amplification cycles:

95°C - 15 sec,

55°C-45 sec,

72°C - 30 sec;

3. final elongation: 72°C for 5 minutes.

During the preparation of the material for the detection of SARS-CoV-2 virus RNA by the method of pre-amplification combined with reverse transcription, the model RNA synthesized on the pGEM-T-SARS-CoV-2 matrix is titrated by preparing serial dilutions (Table 3. Serial dilutions model RNA template samples containing a fragment of the SARS-CoV-2 virus genome).

To assess the efficiency of pre-amplification, the resulting products corresponding to a fragment of the genome of the SARS-CoV-2 virus are visualized using agarose gel electrophoresis (Fig. 1).

The material prepared by the described method without preliminary purification is used for experiments on the detection of SARS-CoV-2 virus RNA using LbCpf1 ribonucleoprotein complexes from Lachnospiraceae containing guide RNAs with unique sequences SEQ ID NO: 1-5.

EXAMPLE 3: PRODUCTION OF GUIDE RNA AND CREATION OF RIBONUCLEOPROTEIN COMPLEXES FOR SARS-COV-2 VIRUS RNA DETECTION

To obtain guide RNAs for the detection of SARS-CoV-2 virus RNA, a set of specific oligonucleotides has been developed, shown in Table 4. Specific oligonucleotides for obtaining a PCR product encoding guide RNAs specific for SARS-CoV-2 virus RNA.

Obtaining guide RNA is carried out in several stages:

1. obtaining a PCR product using a set of specific oligonucleotides (Table 4. Specific oligonucleotides for obtaining a PCR product encoding guide RNAs specific to SARS-CoV-2 virus RNA) encoding a guide RNA capable of binding to a target fragment of the SARS virus genome -CoV-2 containing a hairpin RNA that is recognized by the RNA-directed DNA endonuclease LbCpf1 from Lachnospiraceae;

2. purification of a PCR product encoding a guide RNA specific to a fragment of the genome of the SARS-CoV-2 virus;

3. synthesis of a guide RNA specific to a fragment of the genome of the SARS-CoV-2 virus;

4. Purification of a guide RNA specific to a fragment of the SARS-CoV-2 virus genome.

The NDP product encoding SARS-CoV-2 crRNA no. The size of the amplified fragment encoding crRNA S ARS-CoV-2 No. 26333 is 62 bp.

The PCR product encoding SARS-CoV-2 crRNA guide RNA no. 26413 was obtained in an amplification reaction using PCR mixture-2 blue (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) and specific oligonucleotides T7pr and crRNA 26413 (GenTerra, Russia). The size of the amplified fragment encoding SARS-CoV-2 crRNA No. 26413 is 62 bp.

The PCR product encoding SARS-CoV-2 crRNA guide RNA No. 26432 was obtained in an amplification reaction using PCR mixture-2 blue (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) and specific oligonucleotides T7pr and crRNA 26432 (GenTerra, Russia). The size of the amplified fragment encoding crRNA S ARS-CoV-2 No. 26432 is 62 bp.

The PCR product encoding SARS-CoV-2 crRNA guide RNA no. 26451 was obtained in an amplification reaction using PCR mixture-2 blue (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) and specific oligonucleotides T7pr and crRNA 26451 (GenTerra, Russia). The size of the amplified fragment encoding SARS-CoV-2 crRNA No. 26451 is 62 bp.

The PCR product encoding SARS-CoV-2 crRNA no. The size of the amplified fragment encoding crRNA S ARS-CoV-2 No. 26460 is 62 bp.

Amplification temperature profile for obtaining PCR products encoding guide RNAs containing an RNA hairpin that is recognized by the RNA-guided DNA endonuclease LbCpf1 from Lachnospiraceae:

1. denaturation: 95°C for 3 minutes;

2. 35 amplification cycles:

95°С 15 sec,

55°C-45 sec,

72°C - 30 sec;

3. final elongation: 72°C for 5 minutes.

PCR products encoding guide RNAs specific for a fragment of the SARS-CoV-2 virus genome are visualized by agarose gel electrophoresis.

Purification of PCR products encoding guide RNAs specific to a fragment of the SARS-CoV-2 virus genome is carried out using a commercially available ISOLATE II PCR and Gel Kit (BioLine, USA) according to the manufacturer's instructions. Purified PCR products encoding guide RNAs specific for a fragment of the SARS-CoV-2 virus genome are used as a template for guide RNA synthesis.

Synthesis of guide RNAs specific to a fragment of the SARS-CoV-2 virus genome is carried out by in vitro transcription using commercially available reagent kits (HiScribe™ T7 High Yield RNA Synthesis Kit, NEB, USA) according to the manufacturer's instructions. The reaction products of in vitro transcription are reprecipitated from the reaction mixture by adding sodium chloride to a final concentration of 400 mM and an equal volume of isopropyl alcohol. Such modifications of the manufacturer's protocol introduced by the authors allow increasing the yield of the reaction product and obtaining the desired concentration of the final guide RNA preparation.

The creation of ready-made ribonucleoprotein complexes containing the CRISPR/Cas LbCpfl family protein from Lachnospiraceae and the corresponding guide RNA is carried out by the authors according to a standard protocol with some modifications (w vitro enzymology of Cas9 // Anders C, Jinek M. Methods Enzymol. 2014;546:1- 20. doi: 10.1016/B978-0-12-801185-0.00001-5).

Immediately before combining with the Cas protein, the guide RNA preparation (250 ng) is heated at 90°C for 5 minutes and allowed to cool slowly to room temperature (incubation at room temperature for at least 10 minutes). Such heating is necessary for the formation of the correct hairpin conformation contained in the guide RNA. Many manufacturers of commercial Cas protein preparations skip this step when preparing the ribonucleoprotein complex.

To form the finished ribonucleoprotein complex, 250 ng of the LbCpf1 Cas protein from Lachnospiraceae and the prepared appropriate guide RNA are mixed and incubated for 15 minutes at room temperature. The ribonucleoprotein complex obtained in this way is ready for detection of fragments of the SARS-CoV-2 virus genome.

EXAMPLE 4: DETECTION OF SINGLE COPIES OF SARS-COV-2 VIRUS RNA USING CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES ON THE EXAMPLE OF A MODEL MATRIX

The pre-amplified material obtained by the method described in Example 2 is used as a template for the detection of SARS-CoV-2 virus RNA using CRISPR/Cas ribonucleoprotein complexes obtained by the method described in Example 3.

To detect SARS-CoV-2 virus RNA using CRISPR/Cas ribonucleoprotein complexes containing LbCpfl from Lachnospiraceae, prepare a reaction mixture containing the following components:

10х буфер (100 тМ TrisHCl рН 8,0, 1 М NaCl);

10x buffer (100 mM TrisHCl pH 8.0, 1 M NaCl);

50 mM MgC12 (конечная концентрация в реакционной смеси 10 mM);

50 mM MgC12 (final concentration in the reaction mixture 10 mM);

250 нг рибонуклеопротеинового комплекса (LbCpfl из Lachnospiraceae и направляющие РНК crRNA SARS-CoV-2 №26333 или crRNA SARS-CoV-2 №26413 или crRNA SARS-CoV-2 №26432 или crRNA SARS-CoV-2 №26451 или crRNA SARS-CoV-2 №26460);

250 ng ribonucleoprotein complex (LbCpfl from Lachnospiraceae and SARS-CoV-2 crRNA #26333 or SARS-CoV-2 crRNA #26413 or SARS-CoV-2 crRNA #26432 or SARS-CoV-2 crRNA #26451 or SARS- CoV-2 #26460);

10 pmol флуоресцентный зонд (FAM-TTA-TT-BHQ1);

10 pmol fluorescent probe (FAM-TTA-TT-BHQ1);

Мишень (предварительно амплифицированный фрагмент генома вируса вируса SARS-CoV-2);

Target (preliminarily amplified fragment of the SARS-CoV-2 virus genome);

вода mQ.

mQ water.

The reaction mixtures containing all the necessary components are placed in a QuantStudio 5 cycler (Thermo Fisher Scientific, USA) and the following reaction parameters are set:

60 cycles:

37°С - 35 sec,

37°C - 25 sec, fluorescence recording.

First of all, experiments were carried out to detect single copies of SARS-CoV-2 virus RNA using CRISPR/Cas ribonucleoprotein complexes formed on the basis of LbCpf1 from Lachnospiraceae, using model RNA synthesized on the basis of pGEM-T-SARS plasmid DNA as a target. -CoV-2, containing in its composition a fragment of the genome of the SARS-CoV-2 virus with a size of 260 bp. CRISPR/Cas ribonucleoprotein complexes have been shown to be able to detect single copies of SARS-CoV-2 virus RNA. Typical assay results are shown using examples of real-time fluorescence profiles of pre-amplified fragments treated with ribonucleoportein complexes containing SARS-CoV-2 crRNA #26333 guide RNA or SARS-CoV-2 crRNA #26413 or SARS-CoV-2 crRNA #26432 or SARS-CoV-2 crRNA #26451 or SARS-CoV-2 crRNA #26460 and LbCpfl protein, FIG. 2-6 respectively. Note that the value of the signal obtained during the detection of single copies (1.64 copies/reaction) of SARS-CoV-2 virus RNA exceeded the “noise” value (nonspecific fluorescence of the control sample that did not contain the target sequence) by more than 5 times on average at the 10th analysis cycle (10 minutes), and more than 50 times on average at the 25th analysis cycle (25 minutes).

In the course of the work, the efficiency of detecting single copies of the SARS-CoV-2 virus genome contained in the model RNA matrix was evaluated using various guide RNAs (Fig. 7). It has been shown that CRISPR/Cas ribonucleoprotein complexes formed on the basis of LbCpfl from Lachnospiraceae and guide RNAs detect single copies of the SARS-CoV-2 virus genome with different efficiency, and they can be arranged in the following order of decreasing activity: SARS-CoV- crRNA 2 #26413>crRNA SARS-CoV-2 #26333>crRNA SARS-CoV-2 #26432>crRNA SARS-CoV-2 #26451 ≈ crRNA SARS-CoV-2 #26460.

EXAMPLE 5: DETECTION OF SINGLE COPIES OF SARS-COV-2 VIRUS RNA USING CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES IN A LIMITED PANEL OF CLINICAL SPECIMENS

The developed guide RNAs were tested on a limited panel of clinical samples (10 pcs.) containing the SARS-CoV-2 virus (previously confirmed by real-time PCR).

To detect single copies of SARS-CoV-2 virus RNA using CRISPR/Cas ribonucleoprotein complexes formed on the basis of LbCpf1 from Lachnospiraceae and corresponding guide RNAs, preliminary amplification of SARS-CoV-2 virus genome fragments was carried out. For preliminary amplification, RNA preparations were isolated from clinical samples of 10 patients using a commercially available RIBO-prep kit (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) according to the manufacturer's instructions.

Amplification products encoding fragments of the genome of the SARS-CoV-2 virus are obtained in the amplification reaction as described in Example 2, using the described temperature profile and duration of the amplification reaction.

To assess the efficiency of pre-amplification, the resulting products corresponding to a fragment of the genome of the SARS-CoV-2 virus are visualized using agarose gel electrophoresis (Fig. 8).

The material obtained in this way is used as a template for the detection of single copies of SARS-CoV-2 virus RNA using CRISPR/Cas ribonucleoprotein complexes obtained by the method described in Example 3.

Detection of single copies of SARS-CoV-2 virus RNA using CRISPR/Cas ribonucleoprotein complexes is carried out by the method described in Example 4.

The analysis showed that the CRISPR/Cas ribonucleoprotein complexes formed on the basis of LbCpf1 from Lachnospiraceae and the corresponding guide RNAs have the ability to detect single copies (1.64 copies/reaction) of the SARS-CoV-2 virus in RNA preparations isolated from clinical samples. It is worth noting that the value of the signal obtained during the detection of single copies (1.64 copies/reaction) of SARS-CoV-2 virus RNA using CRISPR/Cas ribonucleoprotein complexes formed on the basis of LbCpf1 from Lachnospiraceae and SARS-CoV- crRNA guide RNAs 2 No. 26333, SARS-CoV-2 crRNA No. 26413 and SARS-CoV-2 crRNA No. 26432, exceeded the “noise” value (non-specific fluorescence of the control sample that does not contain the target sequence) by more than 5 times on average at the 23rd analysis cycle (23 minutes) and more than 50 times on average at the 40th cycle (40 minutes). The value of the signal obtained during the detection of single copies (1.64 copies/reaction) of SARS-CoV-2 virus RNA using CRISPR/Cas ribonucleoprotein complexes formed on the basis of LbCpfl from Lachnospiraceae and SARS-CoV-2 crRNA guide RNA No. 26451 and crRNA SARS-CoV-2 No. 26460, exceeded the “noise” value (non-specific fluorescence of a control sample that did not contain a target sequence) by more than 5 times on average at the 37th analysis cycle (37 minutes) and by more than 50 times in average on the 48th cycle (48 minutes). On FIG. Figure 9 shows typical assay results using examples of endpoint fluorescence values (60 assay cycle, 60 minutes) for pre-amplified targets of SARS-CoV-2 virus genome fragments (10 independent clinical specimens) treated with ribonucleoportein complexes containing SARS-CoV crRNA guide RNAs. -2 #26333 or SARS-CoV-2 crRNA #26413 or SARS-CoV-2 crRNA #26432 or SARS-CoV-2 crRNA #26451 or SARS-CoV-2 crRNA #26460 and LbCpf1 protein.

In conclusion, experiments were carried out to detect SARS-CoV-2 virus RNA in serial dilutions of an RNA preparation isolated from a patient's clinical sample. For this, serial tenfold dilutions of the RNA preparation were prepared from 1:10 to 1:100,000,000 (Table 5. Serial dilutions of SARS-CoV-2 virus RNA isolated from a clinical sample).

Amplification products encoding fragments of the genome of the SARS-CoV-2 virus are obtained in the amplification reaction as described in Example 2, using the described temperature profile and duration of the amplification reaction.

To assess the efficiency of pre-amplification, the resulting products corresponding to a fragment of the genome of the SARS-CoV-2 virus, visualized using agarose gel electrophoresis (Fig. 10).

The material obtained in this way is used as a template for the detection of single copies of SARS-CoV-2 virus RNA using CRISPR/Cas ribonucleoprotein complexes obtained by the method described in Example 3.

Detection of SARS-CoV-2 virus RNA using CRISPR/Cas ribonucleoprotein complexes is carried out by the method described in Example 4.

The analysis showed that CRISPR/Cas ribonucleoprotein complexes formed on the basis of LbCpf1 from Lachnospiraceae and corresponding guide RNAs are able to detect SARS-CoV-2 virus RNA in an RNA preparation diluted 100,000 times. At the same time, when using the guide RNA crRNA SARS-CoV-2 No. 26333, which has the highest efficiency, the signal value exceeded the “noise” value (nonspecific fluorescence of the control sample that did not contain the target) by 49 times. On FIG. Figure 11 shows typical assay results using examples of endpoint fluorescence values (60 assay cycle, 60 minutes) for pre-amplified targets of SARS-CoV-2 virus genome fragments (ten-fold dilutions of an RNA preparation isolated from a patient clinical specimen) treated with ribonucleoportein complexes containing guide RNA SARS-CoV-2 crRNA #26333 or SARS-CoV-2 crRNA #26413 or SARS-CoV-2 crRNA #26432 or SARS-CoV-2 crRNA #26451 or SARS-CoV-2 crRNA #26460 and LbCpfl protein.

Thus, the developed guide RNAs make it possible, as part of the CRISPR/Cas ribonucleoprotein complexes of the CRISPR-Cas12 systems, to detect ultrasensitively single copies of SARS-CoV-2 virus RNA in the reaction after preliminary amplification in RNA preparations (including those diluted in 100,000) isolated from clinical trials. samples containing the SARS-CoV-2 virus.

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Sequence listing

<110> Central Research Institute of Epidemiology of Rospotrebnadzor

<120>

<160> NUMBER OF SEQ ID NO: NOS: 9

<210> SEQ ID NO: NO 1

<211> 40

<212>RNA

<213> artificial

<400> SEQUENCE 1 (crRNA SARS-CoV-2 #26333):

aau uuc uac uaa gug uag aug ugg uau ucu ugc uag uua c 40

<210> SEQ ID NO: NO 2

<211> 40

<212>RNA

<213> artificial

<400> SEQUENCE 2 (crRNA SARS-CoV-2 #26413):

aau uuc uac uaa gug uag auu aac acg aga gua aac gua a 40

<210> SEQ ID NO: NO 3

<211> 40

<212>RNA

<213> artificial

<400> SEQUENCE 3 (crRNA SARS-CoV-2 #26432):

aau uuc uac uaa gug uag auu acg uuu acu cuc gug uua a 40

<210> SEQ ID NO: NO 4

<211> 40

<212>RNA

<213> artificial

<400> SEQUENCE 4 (crRNA SARS-CoV-2 #26451):

aau uuc uac uaa gug uag aug acc aga aga uca gga acu c 40

<210> SEQ ID NO: NO 5

<211> 40

<212>RNA

<213> artificial

<400> SEQUENCE 5 (crRNA SARS-CoV-2 #26460):

aau uuc uac uaa gug uag aug uuc guu uag acc aga aga u 40

<210> SEQ ID NO: NO 6

<211> 32

<212> DNA

<213> artificial

<400> SEQUENCE 6 (E-pr_For1):

gga aga gac agg tac gtt aat agt taa tag cg 32

<210> SEQ ID NO: NO 7

<211> 37

<212> DNA

<213> artificial

<400> SEQUENCE 7 (E-pr_Rev1):

gct aaa att aaa gtt cca aac aga aaa act aat ata a 37

<210> SEQ ID NO: NO 8

<211> 34

<212> DNA

<213> artificial

<400> SEQUENCE 8 (E-pr_For2):

cgt taa tag tta ata gcg tac ttc ttt ttc ttg c 34

<210> SEQ ID NO: NO 9

<211> 34

<212> DNA

<213> artificial

<400> SEQUENCE 9 (E-pr_Rev2):

gct aaa att aaa gtt cca aac aga aaa act aat a 34

<---

Claims (9)

1. A method for detecting SARS-CoV-2 virus RNA, containing: i) conducting pre-amplification of the sample material from a patient suspected of containing the SARS-CoV-2 virus, using one or more specific oligonucleotides selected from SEQ ID NO: 6-9, in order to obtain a target - a pre-amplified fragment of the SARS-CoV virus genome -2, ii) preparing a detection reaction mixture containing the target obtained in step (i); a ribonucleoprotein complex of the CRISPR/Cas system formed from the RNA-guided DNA endonuclease LbCpf1 from Lachnospiraceae and at least one guide RNA with SEQ ID NO:1-5; fluorescent probe and detection buffer, iii) carrying out 30-60 detection cycles in the reaction mixture obtained in step (ii) in a cycler, thus detecting the RNA of the SARS-CoV-2 virus. 2. The method according to p. 1, where the pre-amplified fragment of the SARS-CoV-2 virus genome is represented by a 260 bp fragment. 3. The method according to claim 1 or 2, where the method provides for the detection of SARS-CoV-2 virus RNA at ultra-low concentrations, down to single copies. 4. Specific oligonucleotide selected from SEQ ID NO: 6-9, for use in the method according to paragraphs. 1, 2 or 3 for preliminary amplification of highly conserved fragments of the SARS-CoV-2 virus genome that are recognized by the ribonucleoprotein complex. 5. Kit for use in the method for detecting SARS-CoV-2 virus RNA according to paragraphs. 1, 2 or 3, containing one or more specific oligonucleotides according to claim 4, a ribonucleoprotein complex of the CRISPR/Cas system formed from the RNA-guided DNA endonuclease LbCpf1 from Lachnospiraceae, and at least one guide RNA with SEQ ID NO: 1- 5; fluorescent probe, detection buffer and instructions for use.

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