RU2743861C1 - Crispr-cas system for detecting the antibiotic resistance gene blavim-2 (metallo-beta-lactamase class b vim-2) pseudomonas aeruginosa at ultra-low concentrations - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, namely to a molecule of the guide RNA system CRISPR/CAS. Also disclosed a ribonucleoprotein complex of the CRISPR/CAS system for detecting the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa, formed from the RNA directed DNA endonuclease LbCpf1 from Lachnospiraceae and at least one of the above targeting RNAs. The invention also relates to a kit containing the above molecule or the above complex.

EFFECT: provides detection of single copies of the antibiotic resistance gene blaVIM-2 Pseudomonas aeruginosa.

7 cl, 13 dwg, 5 tbl, 5 ex

Description

Technology area

The invention relates to the field of genetic engineering and biotechnology, namely to the number of means - guide RNAs that can be used in CRISPR-Casl2 systems as part of ribonucleoprotein complexes for the detection (detection, detection) of the antibiotic resistance gene blaVIM-2 (metal-beta-lactamase class VIM-2) Pseudomonas aeruginosa.

The invention allows in vitro detection of single copies of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa.

The guide RNAs described in this application can be used to detect the antibiotic resistance gene blaVIM-2 from Pseudomonas aeruginosa after specific amplification of a DNA fragment of the blaVIM-2 gene. 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); dick 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 new generation diagnostic systems based on CR1SPR technologies to combat the spread of antibiotic-resistant bacterial pathogens.

State of the art

To solve epidemiological problems of decoding outbreaks of infectious diseases, identifying and identifying the pathogen, as well as detecting specific bacterial genes, it is necessary to develop and introduce modern technologies of molecular epidemiology into the practice of supervisory and monitoring services. One of these technologies is the use of CRISPR / CAS genetic editing elements. 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 unexpected mutations. With in-depth studies in the field of application of the CRISPR / CAS system, it was found that it can be used for delicate diagnostic procedures in identifying the causative agent / s of infection in humans, as well as their genotyping.

In 2018, it was shown that one of the enzymes of the CRISPR system, Casl2, after recognizing its target DNA target, begins to hydrolyze nonspecifically single-stranded as well as double-stranded DNA. This property of Casl2 can be used as an indicator of the presence of a specific target, for example, a viral or bacterial genome.

The researchers used this discovery to create a technological platform for the detection of nucleic acids known as DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter). For the first time, DETECTR was used for the detection and genotyping of the human papillomavirus (HPV). The proposed platform combines the Cas12a nuclease, its guide RNA, specific to the HPV nucleic acid, and a fluorescent reporter molecule. DETECTR technology is used to detect target DNA after pre-amplification (Chen JS, Ma Ε, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna J A. CRISPR-Casl2a target binding unleashes indiscriminate single-stranded DNase activity. Science . 2018 Apr 27; 360 (6387): 436-439).

An equally important application of the CRISPR / CAS system is the identification of bacterial pathogens and the detection of specific bacterial genes. For example, using the SHERLOCK platform (Specific High Sensitivity Enzymatic Reporter UnLOCKing), a number of Escherichia coli and Pseudomonas aeruginosa strains were correctly genotyped with low cross-reactivity. In addition, the SHERLOCK platform has been used to differentiate clinical isolates of Klebsiella pneumoniae with two different antibiotic resistance genes, which opens up significant prospects for the creation of multiplex systems for the simultaneous identification of bacterial pathogens and detection of antibiotic resistance genes in them.

In this regard, it is extremely urgent to develop new effective methods for detecting antibiotic resistance genes in bacterial pathogens based on genetic technologies such as CRISPR / CAS.

During the study of the prior art, scientific articles were taken into account describing the development and production of guide RNAs for detecting antibiotic resistance genes in bacterial pathogens using CRISPR / CAS technology (Miiller, V., Rajer, F., Frykholm, K., Nyberg, LK, Quaderi, S., Fritzsche, J., Kristiansson, E., Ambjornsson, T., Sandegren, L., Westerlund, F., 2016. Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR / Cas9 in combination with optical DNA mapping Sci Rep. 6 https://doi.org/10.1038/srep37938; Quan, J., Langelier, C, Kuchta, A., Batson, J., Teyssier, N., Lyden, A., Caldera, S., McGeever, Α., Dimitrov, B., King, R., Wilheim, J ,, Murphy, Μ., Ares, LP, Travisano, KA, Sit, R., Amato, R., Mumbengegwi, DR, Smith, JL, Bennett, Α., Gosling, R., Mourani, PM, Calfee, CS, Neff, NF, Chow, ED, Kim, PS, Greenhouse, B., DeRisi, JL, Crawford, ED, 2019 . FLASH: a next-generation CRISPR diagnostic for multiplexed detectio n of antimicrobial resistance sequences. Nucleic Acids Res. 47, e83. https://doi.org/10.1093/nar/gkz418).

The closest analogue of the invention is the solution published in the article https://doi.org/10.1038/srep37938 (Miiller, V., Rajer, F., Frykholm, K., Nyberg, LK, Quaderi, S., Fritzsche, J., Kristiansson, E., Ambjornsson, T., Sandegren, L., Westerlund, F., 2016. Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR / Cas9 in combination with optical DNA mapping Sci. Rep. 6. https : //doi.org/10.1038/srep37938), which is an analysis based on optical DNA mapping of individual plasmids carrying antibiotic resistance genes, bacterial isolates in nanofluid channels, which provides detailed information about these plasmids, including the presence / absence in them antibiotic resistance genes. The described analysis makes it possible to identify antibiotic resistance genes using CRISPR / CAS9 and guide RNAs specific to antibiotic resistance genes (blaCTX-M group 1, blaCTX-M group 9, blaNDM and blaKPC). During the analysis, the CRISPR / CAS9 ribonucleoprotein complex linearizes circular plasmas in the region of the antibiotic resistance gene; the resulting linear DNA molecules are identified using optical DNA mapping.

The proposed analysis only in the future can be applied to samples with a low concentration of DNA - the proposed method describes the analysis with samples containing about 10 copies of plasmid DNA carrying antibiotic resistance genes (60 ng DNA, plasmid DNA size 67 kb - 220 kb), which is its significant drawback. Also, the disadvantages of the described solution are the need to use expensive high-tech equipment (specialized nanofluid biochips, an inverted fluorescence microscope with a magnification of at least 100x), as well as the need for a complex analysis of the data obtained using specialized software.

Based on this, a technical problem arises, consisting in the need to develop and obtain guide RNAs for the detection of single copies of the antibiotic resistance gene blaVIM-2 (metallo-beta-lactamase class B VIM-2) of Pseudomonas aeruginosa in vitro.

Disclosure of the essence.

The proposed technology is promising for a variety of applications, including quantitative determination of DNA / RNA, rapid multiplex expression detection, and other types of sensitive detection, for example, detecting contamination of samples with nucleic acids. The CRISPR / CAS-based technology is a multifunctional, error-resistant DNA detection technology suitable for rapid diagnosis, including infectious diseases, genotyping of infectious agents, and detection of antibiotic resistance genes for 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:

- high sensitivity;

- the possibility of diagnostics at the patient's bedside;

- the ability to conduct diagnostics in the field without the use of specialized high-tech equipment;

- speed and ease of analysis;

- reduced cost of analysis;

- no need to equip the diagnostic laboratory with expensive equipment;

- no need for isolation of nucleic acids of the pathogen.

The invention relates to new means - guide RNAs, which can be used in CRISPR-Casl2 systems for ultrasensitive detection, identification, detection or detection of the antibiotic resistance gene blaVIM-2 Pseudomonas aeruginosa in biological samples.

The technical objective of the proposed invention is the development of new means - guide RNAs that can be used in CRISPR-Casl2 systems with Casl2 proteins, for example, LbCpfl from Lachnospiraceae, for ultrasensitive detection of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa.

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 the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa to single copies of DNA in one reaction. The invention improves the efficiency of detecting the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa from 1-5 × 10 ⁵ to 2-3 × 10 ² copies / ml.

The technical result is achieved due to:

- development of guide RNA molecules that can be used in CRISPR-Cas12 systems for ultrasensitive detection of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa, where these guide RNAs are selected from the sequences SEQ ID NO: 1-5, are able to bind to the target highly conserved regions of the antibiotic resistance gene blaVIM-2 Pseudomonas aeruginosa, contain an RNA hairpin, which is recognized by the RNA-directed DNA endonuclease LbCpfl from Lachnospiraceae, ensuring the detection of single copies of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa.

- the use of RNA-directed DNA endonuclease LbCpfl from Lachnospiraceae, obtained according to the method developed by the authors earlier (invention under RF patent No. 2707542, priority date 03/28/2019, published 11/27/2019), to create ribonucleoprotein complexes (RPK) of the CRISPR / CAS system suitable for detecting the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa at ultra-low concentrations (single copies).

- development of a set of specific oligonucleotides selected from SEQ ID NO: 6 and SEQ ID NO: 7, for preliminary amplification of a fragment of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa.

- optimization of conditions for preliminary amplification of a fragment of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa.

- determining the conditions for ultrasensitive detection of the antibiotic resistance gene blaVIM-2 Pseudomonas aeruginosa and establishing the sequence of the method steps.

A CRISPR / CAS targeting RNA molecule has been proposed that can bind to target highly conserved regions of the blaVIM-2 antibiotic resistance gene of Pseudomonas aeruginosa. The nucleotide sequence of the guide RNA disclosed herein is selected from SEQ ID NO: 1-5.

The guide RNA molecule contains an RNA hairpin, which is recognized by the RNA guided DNA endonuclease LbCpfl from Lachnospiraceae.

The guide RNA molecule ensures the detection of single copies of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa.

The guide RNAs of the present invention correspond to highly conserved fragments of the Pseudomonas aeruginosa blaVIM-2 antibiotic resistance gene. Most preferred are guide RNAs recognized by the RNA directed DNA endonuclease LbCpfl from Lachnospiraceae, characterized by having or containing a nucleotide sequence selected from:

- SEQ ID NO: 1;

- SEQ ID NO: 2;

- SEQ ID NO: 3;

- SEQ ID NO: 4;

- SEQ ID NO: 5;

- or identical to any of them at least 80% - 99, 99%,

- 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 an RNA-guided DNA endonuclease of the CRISPR / CAS LbCpfl system from Lachnospiraceae, suitable for use for detecting the antibiotic resistance gene blaVIM-2 Pseudomonas aeruginosa in ultra-low concentrations copies).

Ribonucleoprotein complex of the CRISPR / CAS system for detecting the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa, formed from RNA-directed DNA endonuclease LbCpfl from Lachnospiraceae, and at least one guide RNA.

The proposed ribonucleoprotein complex of the CRISPR / CAS system (hereinafter referred to as RPK) is suitable for detecting single copies of the blaVIM-2 antibiotic resistance gene of Pseudomonas aeruginosa.

PKK preparations are solutions containing a guide RNA selected from SEQ ID NO: 1-5, combined with a CRISPR / CAS protein (LbCpfl from Lachnospiraceae) or freeze-dried PKK.

The resulting guide RNAs can be used as part of a kit for detecting the antibiotic resistance gene blaVIM-2 Pseudomonas aeruginosa, with instructions for use.

The kit for detecting the antibiotic resistance gene blaVIM-2 from Pseudomonas aeruginosa may contain a ribonucleoprotein complex and instructions for use.

In this case, at least one guide RNA in the kit can be complexed with a CRISPR / CAS protein (LbCpfl from Lachnospiraceae) in one container or separately in different containers.

The kit may further include components for pre-amplifying highly conserved fragments of the Pseudomonas aeruginosa blaVIM-2 antibiotic resistance gene, including one or more specific oligonucleotides selected from SEQ ID NO: 6 and SEQ ID NO: 7.

Specific oligonucleotides for carrying out preliminary amplification of a fragment of the Pseudomonas aeruginosa blaVIM-2 antibiotic resistance gene according to the present invention correspond to the highly conserved region of the Pseudomonas aeruginosa blaVIM-2 antibiotic resistance gene. Most preferred are oligonucleotides characterized by having or containing a nucleotide sequence selected from:

- SEQ ID NO: 6;

- SEQ ID NO: 7;

- or identical to any of them at least 80% - 99.99%,

- or complementary to any of them,

- or hybridizing with any of them under stringent conditions.

The proposed technology makes it possible to determine single copies of the Pseudomonas aeruginosa blaVIM-2 antibiotic resistance gene in biological samples of a patient selected from fluid and / or tissue, presumably containing Pseudomonas aeruginosa. A biological sample can be a sample of blood, serum or blood plasma, blood cells, saliva, sputum, lymphoid tissues, tissues of hematopoietic organs and other biological materials from a patient, which can be used to analyze for the presence of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa. Brief description of the drawings.

FIG. 1. Visualization of a fragment of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa from 37 to 447 bp. (size 411 i.i.) after preliminary amplification by electrophoresis in agarose gel, where numbers 1-8 indicate:

1 - PCR product blaVIM-2, obtained during the amplification of 1 ng of the model template pGEM-T-blaVIM-2;

2 - PCR product blaVIM-2, obtained during the amplification of 0.1 ng of the model template pGEM-T-blaVIM-2;

3 - PCR product blaVIM-2, obtained during the amplification of 0.01 ng of the model matrix pGEM-T-blaVIM-2;

4 - PCR product blaVIM-2, obtained during the amplification of 0.001 ng of the model template pGEM-T-bla VIM-2;

5 - PCR product blaVIM-2, obtained during the amplification of 0.0001 ng of the pGEM-T-blaVIM-2 model template;

6 - PCR product blaVIM-2, obtained during the amplification of 0.00001 ng of the pGEM-T-blaVIM-2 model template;

7 - negative control that does not contain the pGEM-T-blaVIM-2 model matrix;

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

FIG. 2. Visualization of PCR products encoding guide RNAs specific to the antibiotic resistance gene blaVIM-2 Pseudomonas aeruginosa using agarose gel electrophoresis, where numbers 1-5 indicate:

1 - PCR product encoding sgRNA blaVIM-2 # 93;

2 - PCR product encoding sgRNA blaVIM-2 No. 95;

3 - PCR product encoding sgRNA blaVIM-2 # 207;

4 - PCR product encoding sgRNA blaVIM-2 # 285;

5 - PCR product encoding sgRNA blaVIM-2 # 366;

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

FIG. 3. Profile of fluorescence in real time for pre-amplified target blaVIM-2 Pseudomonas aeruginosa treated with ribonucleoportein complex containing guide RNA sgRNA blaVIM-2 No. 93 and protein LbCpfl.

FIG. 4. Real-time fluorescence profile for a pre-amplified Pseudomonas aeruginosa blaVIM-2 target treated with a ribonucleoportein complex containing sgRNA blaVIM-2 guide RNA # 95 and LbCpfl protein.

FIG. 5. Real-time fluorescence profile for a pre-amplified Pseudomonas aeruginosa blaVIM-2 target treated with a ribonucleoportein complex containing sgRNA blaVIM-2 guide RNA # 207 and LbCpfl protein.

FIG. 6. Real-time fluorescence profile for a pre-amplified Pseudomonas aeruginosa blaVIM-2 target treated with a ribonucleoportein complex containing sgRNA blaVIM-2 guide RNA # 285 and LbCpfl protein.

FIG. 7. Real-time fluorescence profile for pre-amplified Pseudomonas aeruginosa blaVIM-2 target treated with ribonucleoportein complex containing sgRNA blaVIM-2 guide RNA # 366 and LbCpfl protein.

FIG. 8. Endpoint fluorescence values (30 assay cycle, 30 minutes) for pre-amplified Pseudomonas aeruginosa blaVIM-2 target treated with ribonucleoportein complex containing guide RNA sgRNA blaVIM-2 No. 93, sgRNA blaVIM-2 No. 95, sgRNA blaVIM # 207, sgRNA blaVIM-2 # 285 and sgRNA blaVIM-2 # 366.

FIG. 9. Endpoint fluorescence values (30 assay cycle, 30 minutes) for Pseudomonas aeruginosa blaVIM-2 targets pre-amplified from clinical samples treated with ribonucleoportein complex containing sgRNA blaVIM-2 guide RNA # 93.

FIG. 10. Endpoint fluorescence values (30 analysis cycle, 30 minutes) for Pseudomonas aeruginosa blaVIM-2 targets pre-amplified from clinical samples treated with ribonucleoportein complex containing sgRNA blaVIM-2 # 95 guide RNA.

FIG. 11. Endpoint fluorescence values (30 analysis cycle, 30 minutes) for Pseudomonas aeruginosa blaVIM-2 targets pre-amplified from clinical samples, treated with ribonucleoportein complex containing sgRNA blaVIM-2 guide RNA No. 207.

FIG. 12. Endpoint fluorescence values (30 assay cycle, 30 minutes) for Pseudomonas aeruginosa blaVIM-2 targets pre-amplified from clinical samples and treated with ribonucleoportein complex containing sgRNA blaVIM-2 # 285 guide RNA.

FIG. 13. Endpoint fluorescence values (30 assay cycle, 30 minutes) for Pseudomonas aeruginosa blaVIM-2 targets pre-amplified from clinical samples treated with ribonucleoportein complex containing sgRNA blaVIM-2 guide RNA # 366.

Examples of implementation of the invention

EXAMPLE 1: SELECTION OF TARGET SEQUENCES IN THE ANTIBIOTIC RESISTANCE GENE BLAVIM-2 PSEUDOMONAS AERUGINOSA FOR THE CREATION OF GUIDING RNA

To select target sequences in the blaVIM-2 antibiotic resistance gene of Pseudomonas aeruginosa to create guide RNAs, modern algorithms for in silico analysis of nucleotide sequences and publicly available programs were used, including Benchling (https://www.benchling.com/molecular-biology/ ). A list of regions in the Pseudomonas aeruginosa blaVIM-2 antibiotic resistance gene with a theoretically calculated probability of their cleavage in highly conserved regions was compiled (Table 1).

Guide RNAs that specifically recognize the highly conserved antibiotic resistance site of blaVIM-2 Pseudomonas aeruginosa are represented by the unique sequences SEQ ID NO: 1-5.

EXAMPLE 2: PREPARATION OF MATERIAL FOR DETECTION OF ANTIBIOTIC RESISTANCE GENE BLAVIM-2 PSEUDOMONAS AERUGINOSA BY THE METHOD OF PRE-AMPLIFICATION

Preparation of material for the detection of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa is carried out by the method of preliminary amplification. Plasmid DNA pGEM-T-blaVIM-2, which contains a fragment of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa from 37 to 447 bp, is used as a model matrix of the blaVIM-2 antibiotic resistance gene of Pseudomonas aeruginosa of a biological sample. (size 411 bp).

Preliminary amplification of the region corresponding to the fragment of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa from 37 to 447 bp is carried out using specific oligonucleotides with SEQ ID NO: 6-7 shown in Table 2.

A PCR product encoding a fragment of the antibiotic resistance blaVIM-2 of Pseudomonas aeruginosa is obtained in an amplification reaction using PCR mixture-2 blue (FBSI Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) and specific oligonucleotides For blaVIM-2 and Rev blaVIM-2 (GenTerra, Russia) ... The size of the amplified blaVIM-2 fragment is 411 base pairs.

Temperature profile of amplification for obtaining a PCR product, a fragment of the antibiotic resistance gene blaVIM-2 Pseudomonas aeruginosa from 37 to 447 bp:

1.denaturation: 95 ° C for 3 minutes;

2.40 amplification cycles: 95 ° С - 15 sec, 55 ° С - 45 sec, 72 ° С - 30 sec;

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

During the preparation of material for the detection of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa by the method of preliminary amplification, titration of the model matrix pGEM-T-blaVIM-2 is carried out by preparing serial dilutions (Table 3).

To evaluate the efficiency of preliminary amplification, the obtained fragments of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa are visualized by agarose gel electrophoresis (Fig. 1).

The material prepared by the described method is used for experiments to identify the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa using ribonucleoprotein complexes LbCpf1 from Lachnospiraceae containing guide RNA sgRNA blaVIM-2 No. 93, sgRNA blaVIM-2 No. blaVIM-2 No. blaVIM-2 -2 # 285 and sgRNA blaVIM-2 # 366, without preliminary purification.

EXAMPLE 3: OBTAINING GUIDING RNA AND ESTABLISHING RIBONUCLEOPROTEIN COMPLEXES FOR DETECTING ANTIBIOTIC RESISTANCE GENE BLAVIM-2 PSEUDOMONAS AERUGINOSA

To obtain guide RNAs for detecting the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa, a set of specific oligonucleotides was developed, shown in Table 4.

The production of guide RNAs is carried out in several stages:

1.Production of a PCR product using a set of specific oligonucleotides (Table 4) encoding a guide RNA capable of binding to the target highly conserved region of the blaVIM-2 antibiotic resistance gene of Pseudomonas aeruginosa, containing an RNA hairpin that is recognized by RNA-directed DNA endonuclease LbCpfl from Lachnospiraceae;

2. purification of the PCR product encoding a guide RNA specific to a fragment of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa;

3. synthesis of a guide RNA specific to a fragment of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa;

4. Purification of guide RNA specific to a fragment of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa.

The PCR product encoding the guide RNA sgRNA blaVIM-2 No. 93 is obtained in an amplification reaction using PCR-mixture-2 blue (FBSI Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) and specific oligonucleotides T7rg and sgRNA-93-Rev (GenTerra, Russia). The size of the amplified fragment encoding sgRNA blaVIM-2 # 93 is 62 base pairs.

The PCR product encoding the guide RNA sgRNA blaVIM-2 No. 95 is obtained in an amplification reaction using PCR mixture-2 blue (FBSI Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) and specific oligonucleotides T7rg and sgRNA-95-Rev (GenTerra, Russia). The size of the amplified fragment encoding sgRNA blaVIM-2 No. 95 is 62 base pairs.

The PCR product encoding the guide RNA sgRNA blaVIM-2 No. 207 is obtained in an amplification reaction using PCR-mixture-2 blue (FBSI Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) and specific oligonucleotides T7rg and sgRNA-207-Rev (GenTerra, Russia).

The size of the amplified fragment encoding sgRNA blaVIM-2 # 207 is 62 base pairs.

The PCR product encoding the guide RNA sgRNA blaVIM-2 No. 285 is obtained in an amplification reaction using PCR-mixture-2 blue (FBSI Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) and specific oligonucleotides T7rg and sgRNA-285-Rev (GenTerra, Russia). The size of the amplified fragment encoding sgRNA blaVIM-2 # 285 is 62 base pairs.

The PCR product encoding the guide RNA sgRNA blaVIM-2 No. 366 is obtained in an amplification reaction using PCR mixture-2 blue (FBSI Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) and specific oligonucleotides T7rg and sgRNA-366-Rev (GenTerra, Russia). The size of the amplified fragment encoding sgRNA blaVIM-2 # 366 is 62 base pairs.

Temperature profile of amplification for obtaining PCR products encoding guide RNAs:

1.denaturation: 95 ° C for 3 minutes;

2. 35 amplification cycles: 95 ° С - 15 sec, 55 ° С - 45 sec, 72 ° С - 30 sec;

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

PCR products encoding guide RNAs specific to a fragment of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa were visualized by agarose gel electrophoresis (Fig. 2).

Purification of PCR products encoding guide RNAs specific to a fragment of the blaVIM-2 antibiotic resistance gene of Pseudomonas aeruginosa is carried out using a commercially available ISOLATE II PGR and Gel Kit (BioLine, USA) according to the manufacturer's instructions. Purified PCR products encoding guide RNAs specific for a fragment of the blaVIM-2 antibiotic resistance gene of Pseudomonas aeruginosa are used as a template for the synthesis of guide RNAs.

The synthesis of guide RNAs specific to a fragment of the blaVIM-2 antibiotic resistance gene of Pseudomonas aeruginosa 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 products of the in vitro transcription reaction are reprecipitated from the reaction mixture by adding sodium chloride to a final concentration of 400 tM 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 a ready-made ribonucleoprotein complex containing a protein of the CRISPR / CAS LbCpf1 family from Lachnospiraceae and a guide RNA was carried out by the authors according to the standard protocol with some modifications (In 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). This heating is necessary for the formation of the correct conformation of the hairpin 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 guide RNA are mixed and incubated for 15 minutes at room temperature.

The ribonucleoprotein complex obtained in this way is ready for detection of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa.

EXAMPLE 4: DETECTION OF SINGLE COPIES OF ANTIBIOTIC RESISTANCE GENE BLAVIM-2 PSEUDOMONAS AERUGINOSA USING RIBONUCLEOPROTEIN COMPLEXES CRISPR / CAS 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 detecting the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa using ribonucleoirotein complexes CRISPR / CAS obtained by the method described in Example 3.

To detect the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa using ribonucleoprotein complexes CRISPR / CAS, a reaction mixture is prepared containing the following components:

- 10 × buffer (100 mM TrisHCl pH 8.0, 1 Μ NaCl);

- 50 mM MgCT ₂ (final concentration in the reaction mixture 10 mM);

250 ng of ribonucleoprotein complex (LbCpf1 from Lachnospiraceae and guide RNAs sgRNA blaVIM-2 # 93, sgRNA blaVIM-2 # 95, sgRNA blaVIM-2 # 207, sgRNA blaVIM-2 # 285 and sgRNA # 366);

- 20 pmol fluorescent probe (6FAM-TTATT-BHQ1);

- Target (pre-amplified fragment of the antibiotic resistance gene blaVIM-2 Pseudomonas aeruginosa);

- water mQ.

The reaction mixtures containing all the necessary components are placed in a DTPrime 5 thermocycler (DNA-Technology, Russia) and the following reaction parameters are set:

30-60 cycles:

37 ° С-35 sec,

37 ° С - 25 sec, fluorescence shooting.

First of all, experiments were carried out to detect single copies of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa using ribonucleoprotein complexes CRISPR / CAS, formed on the basis of LbCpfl from Lachnospiraceae, using as a target a model matrix - plasmid DNA pVIM-T-bla containing a fragment of the antibiotic resistance gene blaVIM-2 Pseudomonas aeruginosa from 37 to 447 bp. size 411 p.o. It was shown that CRISPR / CAS ribonucleoprotein complexes are capable of detecting single copies of the blaVIM-2 antibiotic resistance gene of Pseudomonas aeruginosa. Typical analysis results are shown by examples of real-time fluorescence profiles for a pre-amplified Pseudomonas aeruginosa blaVIM-2 target treated with ribonucleoportein complexes containing guide RNAs sgRNA blaVIM-2 No. 93, sgRNA blaVIM-2 No. 95, sgRNA No. blaVIM-2 blaVIM-2 # 285 and sgRNA blaVIM-2 # 366 and LbCpfl protein in FIG. 3, FIG. 4, FIG. 5, Fig. 6 and FIG. 7 respectively. Note that already at the 30th cycle of analysis (30 minutes), the value of the signal obtained during the detection of single copies (1.5 copies / reaction) of the blaVIM-2 gene of Pseudomonas aeruginosa exceeded the value of "noise" (nonspecific fluorescence of the control sample, not containing targets) at least three times.

In the course of the work, the efficiency of detecting single copies of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa, contained in the model matrix, was assessed using various guide RNAs. It was shown that ribonucleoprotein complexes CRISPR / CAS, formed on the basis of LbCpfl from Lachnospiraceae and guide RNAs, reveal single copies of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa with different efficiencies, and they can be arranged in the following order of decreasing activity: sgRNA blaVIM 207> sgRNA blaVIM-2 # 93> sgRNA blaVIM-2 # 366> sgRNA blaVIM-2 # 285> sgRNA blaVIM-2 # 95 (Fig. 8).

EXAMPLE 5: DETECTION OF SINGLE COPIES OF ANTIBIOTIC RESISTANCE GENE BLAVIM-2 PSEUDOMONAS AERUGINOSA USING RIBONUCLEOPROTEIN COMPLEXES CRISPR / CAS ON A LIMITED PANEL

The developed guide RNAs were tested on a limited panel of clinical samples (10 pieces) containing Pseudomonas aeruginosa carrying the blaVIM-2 antibiotic resistance gene (previously confirmed by microbiological methods and next generation sequencing).

To detect single copies of the blaVIM-2 antibiotic resistance gene using ribonucleoprotein complexes CRISPR / CAS, preliminary amplification of fragments of this gene was carried out. For preliminary amplification, serial (according to Table 3) dilutions of DNA preparations isolated from clinical samples of 10 patients using a commercially available DNeasy Blood & Tissue Kit (QIAGEN, USA) were prepared according to the manufacturer's instructions.

PCR products encoding a fragment of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa are obtained in an amplification reaction using PCR-mixture-2 blue (FBSI Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) as described in Example 2, using the described temperature profile and duration of the amplification reaction.

The material obtained in this way is used as a template for the detection of single copies of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa using ribonucleoprotein complexes CRISPR / CAS obtained by the method described in Example 3.

Detection of single copies of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa using ribonucleoprotein complexes CRISPR / CAS is carried out by the method described in Example 4.

The analysis showed that CRISPR / CAS ribonucleoprotein complexes are capable of detecting single copies (1.5 copies / reaction) of the blaVIM-2 antibiotic resistance gene of Pseudomonas aeruginosa in DNA preparations isolated from clinical samples. At the same time, already at 2-15 cycles (2-15 minutes) of the analysis, the signal value exceeded the value of "noise" (nonspecific fluorescence of the control sample containing no target) by three times, and by the 5-26 cycle (5-26 minutes) of the analysis - more than 5 times (wide range and difference in cycles are due to differences in guide RNAs used in the analysis, Table 5).

Typical assay results are shown by examples of fluorescence endpoint values (30 assay cycle, 30 minutes) for pre-amplified blaVTM-2 Pseudomonas aeruginosa targets (10 independent clinical samples) treated with ribonucleoportein complexes containing sgRNA blaVIM-2 guide RNAs # 93, sgRNA blaVIM-2 # 95, sgRNA blaVIM-2 # 207, sgRNA blaVIM-2 # 285 and sgRNA blaVIM-2 # 366 and LbCpfl protein, in FIG. 9, Fig. 10, Fig. 11, Fig. 12 and FIG. 13 respectively.

The efficiency of detecting single copies of the Pseudomonas aeruginosa blaVIM-2 antibiotic resistance gene contained in DNA preparations isolated from clinical samples using various guide RNAs in the CRISPR / CAS ribonucleoprotein complexes formed on the basis of LbCpfl from Lachnospiraceae can be presented in the following descending order : sgRNA blaVIM-2 # 207> sgRNA blaVIM-2 # 93> sgRNA blaVIM-2 # 285> sgRNA blaVIM-2 # 366> sgRNA blaVIM-2 # 95 (Table 5).

Thus, the developed guide RNAs allow ultrasensitive detection of single copies of the blaVIM-2 antibiotic resistance gene of Pseudomonas aeruginosa in clinical samples after preliminary amplification in the CRISPR / CAS ribonucleoprotein complexes.

Claims (14)

1. Molecule of the CRISPR / CAS system guide RNA, which is able to bind to the target highly conserved regions of the antibiotic resistance gene blaVIM-2 (metallo-beta-lactamase class B VIM-2) of Pseudomonas aeruginosa, where the nucleotide sequence of the guide RNA is selected from the sequences of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5. 2. A guide RNA molecule according to claim 1, characterized in that it contains an RNA hairpin, which is recognized by the RNA directed DNA endonuclease LbCpf1 from Lachnospiraceae. 3. A guide RNA molecule according to claim 1 or 2, characterized in that it provides detection of single copies of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa. 4. Ribonucleoprotein complex of the CRISPR / CAS system for detecting the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa, formed from RNA-directed DNA endonuclease LbCpf1 from Lachnospiraceae and at least one of the targeting RNAs according to any one of paragraphs. 1-3. 5. The ribonucleoprotein complex of the CRISPR / CAS system according to claim 4, suitable for detecting single copies of the antibiotic resistance gene blaVIM-2 of Pseudomonas aeruginosa. 6. Kit for detecting the antibiotic resistance gene blaVIM-2 Pseudomonas aeruginosa, containing: (i) (a) a CRISPR / CAS guide RNA molecule according to any one of claims. 1-3 and directed DNA endonuclease LbCpf1 from Lachnospiraceae; or (i) (b) a ribonucleoprotein complex according to any one of claims. 4, 5; and (ii) instructions for use. 7. Kit for detecting the antibiotic resistance gene blaVIM-2 Pseudomonas aeruginosa, containing: (i) (a) a CRISPR / CAS guide RNA molecule according to any one of claims. 1-3 and directed DNA endonuclease LbCpf1 from Lachnospiraceae; or (i) (b) a ribonucleoprotein complex according to any one of claims. 4, 5; and (ii) one or more specific oligonucleotides selected from SEQ ID NO: 6 and SEQ ID NO: 7, and (iii) instructions for use.

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