RU2841370C1 - Method of detecting the antibiotic resistance gene bla-oxa-1 in ultra-low concentrations and specific oligonucleotides for use in the method - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, particularly to a method of detecting antibiotic resistance gene bla-OXA-1. Said method involves preliminary amplification of material from a patient using one or more specific oligonucleotides selected from SEQ ID NO: 4-5, in order to obtain a target, preparation of a reaction mixture for detection containing said target, a ribonucleoprotein complex of the CRISPR-Cas system, formed from RNA-targeted DNA endonuclease LbCpf1 from Lachnospiraceae and at least one guide RNA with SEQ ID NO: 1-3; a fluorescent probe and a detection buffer, performing 30-60 detection cycles in said reaction mixture. Invention also relates to a specific oligonucleotide selected from SEQ ID NO: 4-5, as well as a kit for use in said method.

EFFECT: present invention provides a considerable increase in the efficiency of detecting the antibiotic resistance gene bla-OXA-1.

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Description

The invention relates to the field of genetic engineering and biotechnology, namely to a number of RNA guide agents that can be used in CRISPR-Cas 12 systems as part of ribonucleoprotein complexes for identifying (detecting) the antibiotic resistance gene bla-OXA-1, as well as to diagnostic methods and kits.

The invention allows for the in vitro detection of single copies of the antibiotic resistance gene bla-OXA-1.

The guide RNAs described in this application can be used to detect the bla-OXA-1 antibiotic resistance gene after specific amplification of fragments of the bla-OXA-1 antibiotic resistance gene. The amplification can be carried out by various methods, including polymerase chain reaction (PCR); loop-mediated 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 diagnostic methods for infectious diseases.

To solve epidemiological problems of decoding outbreaks of infectious diseases, detecting and identifying the pathogen, and detecting specific bacterial genes, it is necessary to develop and implement modern molecular epidemiology technologies into the practice of surveillance and monitoring services. One of these technologies is the use of elements of genetic editing of the CRISPR-Cas system. This technology is developing quite effectively in relation to the creation of treatments for certain diseases, despite a number of difficulties associated with the occurrence of unforeseen mutations. In-depth studies in the field of application of the CRISPR-Cas system have shown that it can be used for fine diagnostic procedures in identifying the pathogen/s of 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, begins to non-specifically hydrolyze single-stranded DNA. This property of Cas12 can be used as an indicator of the presence of a specific target, for example, the genome of a virus or bacteria. Researchers used this discovery to create a nucleic acid detection technology platform known as DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter). DETECTR was first used to detect and genotype 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. The DETECTR technology is used to detect the target DNA after preliminary amplification [J.S. Chen, E. Ma, L.B. Harrington, M. Da Costa, X. Tian, J.M. Palefsky, J.A. Doudna, CRISPR-Cas 12a target binding releases indiscriminate single-stranded DNase activity, Science 360 (6387) (2018) 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, it was possible to correctly genotype a number of strainsEscherichia coli And Pseudomonas aeruginosaa with low cross-reactivity. In addition, the SHERLOCK platform has been used to differentiate clinical isolatesKlebsiella pneumoniaewith 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, the task of developing new effective methods for identifying antibiotic resistance genes in bacterial pathogens based on genetic technologies such as CRISPR-Cas is extremely urgent.

The state of the art includes scientific articles describing the detection of the antibiotic resistance gene bla-OXA-1 in biological material [Colom K., Perez J., Alonso R., Fernandez-Aranguiz A., Larino E., Cisterna R. Simple and reliable multiplex PCR assay for detection of blaTEM, bla(SHV) and bla-OXA-1 genes in Enterobacteriaceae. FEMS Microbiol Lett. 2003; 223 (2): 147-151. doi:10.1016/S0378-1097(03)00306-9; Ogutu J.O., Zhang Q., Huang Y. et al. Development of a multiplex PCR system and its application in detection of blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and bla-OXA-1 group genes in clinical Klebsiella pneumoniae and Escherichia coli strains. J Antibiot (Tokyo). 2015; 68 (12): 725-733. doi:10.1038/ja.2015.68]. However, the cited publications do not provide information on the sensitivity of the presented methods. There is also a publication describing the detection of related antibiotic resistance genes of the bla-OXA family by PCR, the sensitivity of which is 3.2 copies/reaction [Probst K., Boutin S., Bandilla M., Heeg K., Dalpke A.N. Fast and automated detection of common carbapenemase genes using multiplex real-time PCR on the BD MAX™ system. J Microbiol Methods. 2021;185:106224. doi:10.1016/j.mimet.2021.106224].

Until now, most kits for detection of extended-spectrum beta-lactamases, including beta-lactamase a, encoded by the bla-OXA-1 gene, are based on the agar diffusion test methodology and determination of the isoelectric point, which is generally considered sufficient for identification of strains producing extended-spectrum beta-lactamases. However, due to the emergence of a large number of different bla-SHV, bla-TEM and bla-CTX-M beta-lactamases, isoelectric focusing is apparently not an appropriate method for establishing the phenotype of extended-spectrum beta-lactamases. In addition, the described methods require a long time, specialized equipment, and qualified personnel (microbiology).

The closest analogues of the claimed solution are inventions under patents RU 2734520 (priority date 15.04.2020) aimed at obtaining guide RNAs intended for the development of highly sensitive and high-tech diagnostic systems of a new generation for detecting the antibiotic resistance gene blaVIM-2 (metallo-beta-lactamase class B VIM-2) of Pseudomonas aeruginosa ; RU 2782588 (priority date 27.12.2021), disclosing the technical feasibility of obtaining guide RNAs intended for the development of highly sensitive and high-tech diagnostic systems of a new generation for detecting the antibiotic resistance gene mecA of Staphylococcus aureus based on CRISPR technologies; RU 2791880 (priority date 27.12.2021), which discloses the possibility of obtaining guide RNAs intended for the development of highly sensitive and high-tech next-generation diagnostic systems for the detection of exoU encoding the exotoxin of the type 3 secretion system of Pseudomonas aeruginosa based on CRISPR technologies. However, in order to expand the range of such diagnostic systems and improve methods for diagnosing infectious diseases, epidemiological surveillance and monitoring the spread of antibiotic-resistant microorganisms, it is necessary to develop new methods for identifying antibiotic resistance genes of pathogenic microorganisms.

Based on the above, a technical problem arises, which consists in the need to develop and obtain guide RNAs for the detection of single copies of the antibiotic resistance gene bla-OXA-1, in vitro .

The proposed technology is promising for a variety of applications, including DNA/RNA quantification, rapid multiplex expression detection, and other sensitive detections, such as nucleic acid contamination detection. CRISPR-Cas-based technology is a multifunctional, error-tolerant DNA detection technology suitable for rapid diagnosis, including infectious diseases, and genotyping of infectious agents and detection of antibiotic resistance genes and exotoxin-encoding genes of bacterial pathogens.

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

• high sensitivity;

• the ability to conduct diagnostics at the patient’s bedside;

• the ability to carry out diagnostics in field conditions 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 to isolate the nucleic acids of the pathogen.

The invention relates to new agents - guide RNAs that can be used in CRISPR-Cas 12 systems for ultrasensitive detection, identification, detection or sensing of the antibiotic resistance gene bla-OXA-1 in biological samples.

The technical task of the proposed invention is to develop new means - guide RNAs that can be used in CRISPR-Cas 12 systems with Cas 12 proteins, such as the LbCpfl protein from Lachnospiraceae , for ultrasensitive detection of the antibiotic resistance gene bla-OXA-1.

In the implementation of the present invention, according to the set of essential features given in the invention formula, an unexpected technical result is achieved - the possibility of ultrasensitive detection of the antibiotic resistance gene bla-OXA-1 to single copies of the antibiotic resistance gene bla-OXA-1 in one reaction. The invention provides an increase in the efficiency of detection of the antibiotic resistance gene bla-OXA-1 up to 2.5 times.

The technical result is achieved due to:

• development of guide RNA molecules that can be used in CRISPR-Cas 12 systems for ultra-sensitive detection of the bla-OXA-1 antibiotic resistance gene, wherein said guide RNAs are selected from the sequences of SEQ ID NO: 1-3, are capable of binding to target highly conserved regions of the bla-OXA-1 antibiotic resistance gene, contain a hairpin RNA that is recognized by the RNA-guided DNA endonuclease LbCpfl from Lachnospiraceae , ensuring detection of single copies of the bla-OXA-1 antibiotic resistance gene.

• the use of RNA-guided DNA endonuclease LbCpfl from Lachnospiraceae , obtained according to the method previously developed by the authors (Patent RU 2707542, priority date 03/28/2019), to create ribonucleoprotein complexes (RPCs) of the CRISPR-Cas system suitable for detecting the antibiotic resistance gene bla-OXA-1 in ultra-low concentrations (single copies).

• development of a set of specific oligonucleotides selected from SEQ ID NO: 4-5 for preliminary amplification of a fragment of the antibiotic resistance gene bla-OXA-1.

• optimization of conditions for preliminary amplification of the bla-OXA-1 antibiotic resistance gene fragment.

• determining the conditions for conducting ultrasensitive detection of the antibiotic resistance gene bla-OXA-1 and establishing the sequence of stages of the method.

The guide RNAs according to the present invention correspond to highly conserved fragments of the antibiotic resistance gene bla-OXA-1. Most preferred are guide RNAs recognized by the RNA-guided DNA endonuclease LbCpfl from Lachnospiraceae , characterized by, having or containing a nucleotide sequence selected from:

• SEQ ID NO: l;

• SEQ ID NO: 2;

• SEQ ID NO: 3;

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

•or complementary to any of them,

• or hybridizing with any of them under strict conditions.

A set of specific oligonucleotides for carrying out preliminary amplification of a fragment of the antibiotic resistance gene bla-OXA-1, according to the present invention, correspond to a highly conserved region of the antibiotic resistance gene bla-OXA-1. Most preferred are oligonucleotides characterized by, having or containing a nucleotide sequence selected from:

• SEQ ID NO: 4;

• SEQ ID NO: 5;

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

• or complementary to any of them,

• or hybridizing with any of them under strict conditions.

According to the proposed invention, ribonucleoprotein complexes (RNCs) are obtained, consisting of at least one guide RNA and an RNA-guided DNA nuclease of the CRISPR-Cas system LbCpfl from Lachnospiraceae , suitable for use in detecting the antibiotic resistance gene bla-OXA-1 in ultra-low concentrations (single copies).

The RPC preparations are solutions containing a guide RNA selected from SEQ ID NO: 1-3, combined with a protein of the CRISPR-Cas system (LbCpfl from Lachnospiraceae ) or freeze-dried RPC.

The obtained guide RNAs can be used as part of a kit for detecting the antibiotic resistance gene bla-OXA-1 with instructions for use.

The method for detecting the antibiotic resistance gene bla-OXA-1 involves:

i) performing preliminary amplification of the sample material from the patient, presumably containing the antibiotic resistance gene bla-OXA-1, using one or more specific oligonucleotides selected from SEQ ID NO: 4-5, in order to obtain a target - a pre-amplified fragment of the antibiotic resistance gene bla-OXA-1,

ii) preparing a detection reaction mixture comprising a target obtained in step (i); a ribonucleoprotein complex of the CRISPR-Cas system formed from the RNA-guided DNA endonuclease LbCpfl from Lachnospiraceae and at least one guide RNA with SEQ ID NO: 1-3; a fluorescent probe and a detection buffer,

iii) carrying out 30-60 detection cycles in the reaction mixture obtained in step (ii) in a thermocycler,

with the discovery of the antibiotic resistance gene bla-OXA-1.

The pre-amplified fragment of the antibiotic resistance gene bla-OXA-1, according to the proposed method, can be represented by a 262 bp fragment of the antibiotic resistance gene bla-OXA-1.

The proposed method ensures the detection of the antibiotic resistance gene bla-OXA-1 in ultra-low concentrations, down to single copies.

A specific oligonucleotide for use in the method is selected from the group SEQ ID NO: 4-5 and is used for preliminary amplification of a highly conserved fragment of the antibiotic resistance gene bla-OXA-1, which is recognized by the ribonucleoprotein complex.

A kit for use in a method for detecting the bla-OXA-1 antibiotic resistance gene comprises a ribonucleoprotein complex of the CRISPR-Cas system formed from the RNA-guided DNA endonuclease LbCpfl from Lachnospiraceae and at least one guide RNA with SEQ ID NO: 1-3; a fluorescent probe, a detection buffer and instructions for use.

The kit may additionally include components for preliminary amplification of a highly conserved fragment of the bla-OXA-1 antibiotic resistance gene, including one or more specific oligonucleotides selected from SEQ ID NO: 4-5.

In this case, at least one guide RNA in the kit may be in a complex with a protein of the CRISPR-Cas system (LbCpfl from Lachnospiraceae ) in one container or separately in different containers.

The proposed technology allows for the detection of single copies of the bla-OXA-1 antibiotic resistance gene in patient biological samples selected from fluid and/or tissue presumably containing the bla-OXA-1 antibiotic resistance gene. A biological sample may be a sample of blood, serum or blood plasma, blood cells, saliva, sputum, lymphoid tissues, hematopoietic organ tissues and other biological materials from a patient that can be used to analyze for the presence of the bla-OXA-1 antibiotic resistance gene.

Brief description of the drawings

Fig. 1. Visualization of the amplified fragment of the antibiotic resistance gene bla-OXA-1 (262 bp in size) after preliminary amplification using oligonucleotides OXA-1 for 2 and OXA-1 rev 1 using electrophoresis in agarose gel, where the numbers 1-8 indicate:

1 - Product obtained during the amplification of 1.25×10 ^b copies of the model template pGEM-T-bla-OXA-1;

2 - Product obtained during amplification of 1.25×10 ⁵ copies of the model template pGEM-T-bla-OXA-1;

3 - Product obtained during the amplification of 1.25× ¹⁰⁴ copies of the model template pGEM-T-bla-OXA-1;

4 - Product obtained during the amplification of 1250 copies of the model template pGEM-T-bla-OXA-1;

5 - Product obtained during the amplification of 125 copies of the model template pGEM-T-bla-OXA-1;

6 - Product obtained during the amplification of 12.5 copies of the model matrix pGEM-T-bla-OXA-1;

7 - Product obtained during the amplification of 1.25 copies of the model template pGEM-T-bla-OXA-1;

8 - negative control, not containing the model matrix pGEM-T-bla-OXA-1;

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

Fig. 2. Real-time fluorescence profile of a pre-amplified fragment of the antibiotic resistance gene bla-OXA-1 treated with a ribonucleoprotein complex containing the guide RNA crRNA bla-OXA-1 #63 and the LbCpfl protein, which graphically visualizes the detection of a fragment of the model template pGEM-T-bla-OXA-1 by the ribonucleoprotein complexes LbCpfl from Lachnospiraceae containing the guide RNA crRNA bla-OXA-1 #63, where:

- the vertical axis shows the values of the normalized fluorescent signal generated by the dye;

- the horizontal axis shows the fluorescence time, in minutes;

- curves 9-16 show the number of copies of nucleic acid per reaction, namely:

9 - 1,250,000 copies/reaction;

10 - 125,000 copies/reaction;

11 - 12,500 copies/reaction;

12 - 1250 copies/reaction;

13 - 125 copies/reaction;

14 - 12.5 copies/reaction;

15 - 1.25 copies/reaction;

16 - K - (negative control).

Fig. 3. Real-time fluorescence profile of a pre-amplified fragment of the antibiotic resistance gene bla-OXA-1 treated with a ribonucleoprotein complex containing the guide RNA crRNA bla-OXA-1 #185 and the protein LbCpfl, which graphically visualizes the detection of a fragment of the model template pGEM-T-bla-OXA-1 using the ribonucleoprotein complexes LbCpfl from Lachnospiraceae containing the guide RNA crRNA bla-OXA-1 #185, where:

- the vertical axis shows the values of the normalized fluorescent signal generated by the dye;

- the horizontal axis shows the fluorescence time, in minutes;

- curves 17-24 show the number of copies of nucleic acid per reaction, namely:

17 - 1,250,000 copies/reaction;

18 - 125,000 copies/reaction;

19 - 12,500 copies/reaction;

20 - 1250 copies/reaction;

21 - 125 copies/reaction;

22 - 12.5 copies/reaction;

23 - 1.25 copies/reaction;

24 - K - (negative control).

Fig. 4. Real-time fluorescence profile of a pre-amplified fragment of the antibiotic resistance gene bla-OXA-1 treated with a ribonucleoprotein complex containing the guide RNA crRNA bla-OXA-1 #221 and the protein LbCpfl, which graphically visualizes the detection of a fragment of the model template pGEM-T-bla-OXA-1 by the ribonucleoprotein complexes LbCpfl from Lachnospiraceae containing the guide RNA crRNA bla-OXA-1 #221, where:

- the vertical axis shows the values of the normalized fluorescent signal generated by the dye;

- the horizontal axis shows the fluorescence time, in minutes;

- curves 25-32 show the number of copies of nucleic acid per reaction, namely:

25 - 1,250,000 copies/reaction;

26 - 125,000 copies/reaction;

27 - 12,500 copies/reaction;

28 - 1250 copies/reaction;

29 - 125 copies/reaction;

30 - 12.5 copies/reaction;

31 - 1.25 copies/reaction;

32 - K - (negative control).

Fig. 5. End-point fluorescence values (60 analysis cycles, 60 minutes) for a pre-amplified fragment of the antibiotic resistance gene bla-OXA-1 treated with ribonucleoprotein complexes containing guide RNAs crRNA bla-OXA-1 #63, crRNA bla-OXA-1 #185 and crRNA bla-OXA-1 #221, where the graph visualizes the detection of the pGEM-T-bla-OXA-1 model template fragment using LbCpfl ribonucleoprotein complexes from Lachnospiraceae , with:

- the vertical axis shows the values of the normalized fluorescent signal generated by the dye;

- the horizontal axis indicates the number of copies of the model matrix in the reaction;

- column the efficiency of detection of single copies of the pGEM-T-bla-OXA-1 model matrix fragment using crRNA bla-OXA-1 No. 63 was visualized;

- column the efficiency of detection of single copies of the pGEM-T-bla-OXA-1 model matrix fragment using crRNA bla-OXA-1 No. 185 was visualized;

- column The efficiency of detection of single copies of the pGEM-T-bla-OXA-1 model matrix fragment using crRNA bla-OXA-1 No. 221 was visualized.

Fig. 6. Visualization of the bla-OXA-1 antibiotic resistance gene fragment (262 bp in size) amplified from clinical samples after preliminary amplification using OXA-l_for_2 and OXA-1_rev_1 oligonucleotides using electrophoresis in agarose gel.

Fig. 7. End-point fluorescence values (60 assay cycles, 60 minutes) for a pre-amplified fragment of the antibiotic resistance gene bla-OXA-1 from clinical samples treated with ribonucleoprotein complexes containing guide RNAs crRNA bla-OXA-1 #63, crRNA bla-OXA-1 #185 and crRNA bla-OXA-1 #221, where the graph visualizes the detection of the antibiotic resistance gene fragment bla-OXA-1 using the ribonucleoprotein complexes LbCpf1 from Lachnospiraceae on a limited panel of clinical samples, with:

- the vertical axis shows the values of the normalized fluorescent signal generated by the dye;

- sample identifiers are indicated horizontally;

- column the efficiency of detection of the fragment of the antibiotic resistance gene bla-OXA-1 contained in the composition of drugs isolated from clinical samples using crRNA bla-OXA-1 No. 63 was demonstrated;

- column the efficiency of detection of the fragment of the antibiotic resistance gene bla-OXA-1 contained in the composition of drugs isolated from clinical samples using crRNA bla-OXA-1 No. 185 was demonstrated;

- column The efficiency of detection of the fragment of the antibiotic resistance gene bla-OXA-1 contained in the composition of drugs isolated from clinical samples using crRNA bla-OXA-1 No. 221 was demonstrated.

Examples of implementation of the invention

EXAMPLE 1: SELECTION OF TARGET SEQUENCES IN THE BLA-OXA-1 ANTIBIOTIC RESISTANCE GENE TO CREATE GUIDE RNAS

To select target sequences in the bla-OXA-1 antibiotic resistance gene for creating guide RNAs, modern in silico algorithms for nucleotide sequence analysis and open-source programs, including Benchling (https://www.benchling.com/molecular-biology/), were used. A list of regions of the bla-OXA-1 antibiotic resistance gene was compiled with a theoretically calculated probability of their cleavage in highly conserved regions (Table 1). Guide RNAs that specifically recognize highly conserved regions of the bla-OXA-1 antibiotic resistance gene are represented by unique sequences SEQ ID NO: 1-3.

EXAMPLE 2: PREPARATION OF MATERIAL FOR DETECTION OF THE ANTIBIOTIC RESISTANCE GENE BLA-OXA-1 BY THE METHOD OF PRE-AMPLIFICATION

The material for detection of the antibiotic resistance gene bla-OXA-1 was prepared by the preliminary amplification method. Plasmid DNA pGEM-T-bla-OXA-1, containing a fragment of the antibiotic resistance gene bla-OXA-1 of 321 bp, was used as a model matrix containing a fragment of the antibiotic resistance gene bla-OXA-1.

Preliminary amplification of the region corresponding to the fragment of the antibiotic resistance gene bla-OXA-1 was carried out using specific oligonucleotides with SEQ ID NO: 4 and SEQ ID NO: 5.

The product encoding the fragment of the antibiotic resistance gene bla-OXA-1 was obtained by PCR using specific oligonucleotides OXA-l_for_2 and OXA-1_rev_1 (GenTerra, Russia) and TaqF polymerase (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia). The size of the amplified fragment was 262 nucleotide pairs.

Temperature profile of amplification for obtaining PCR products of fragments of the antibiotic resistance gene bla-OXA-1:

1. denaturation: 95°C for 3 minutes;

2. 40 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 the antibiotic resistance gene bla-OXA-1 by the preliminary amplification method, titration of the model matrix pGEM-T-bla-OXA-1 was carried out by preparing serial dilutions (Table 2).

To assess the efficiency of preliminary amplification, the obtained fragment of the antibiotic resistance gene bla-OXA-1 was visualized using electrophoresis in agarose gel (Fig. 1).

The material prepared in the described manner was used for experiments to identify the antibiotic resistance gene bla-OXA-1 using ribonucleoprotein complexes LbCpfl from Lachnospiraceae containing guide RNAs crRNA bla-OXA-1 No. 63, crRNA bla-OXA-1 No. 185 and crRNA bla-OXA-1 No. 221, without preliminary purification.

EXAMPLE 3: CREATION OF RIBONUCLEOPROTEIN COMPLEXES FOR DETECTION OF THE ANTIBIOTIC RESISTANCE GENE BLA-OXA-1

Specific RNA oligonucleotides listed in Table 3 were used as guide RNAs to detect the antibiotic resistance gene bla-OXA-1.

The authors created a ready-made ribonucleoprotein complex containing the CRISPR-Cas 12 LbCpfl protein from Lachnospiraceae and guide RNA according to the standard protocol with some modifications [C. Anders, M. Jinek, In vitro enzymology of Cas9, Methods Enzymol. 546 (2014) 1–20, https://doi.org/10.1016/B978-0-12-801155-0.00001-51]. To form the ready-made ribonucleoprotein complex, 250 ng of the Cas protein LbCpfl from Lachnospiraceae and guide RNA were mixed and incubated for 10 minutes at room temperature. The ribonucleoprotein complex obtained in this way is ready for detection of the antibiotic resistance gene bla-OXA-1.

EXAMPLE 4: DETECTION OF SINGLE COPIES OF THE ANTIBIOTIC RESISTANCE GENE BLA-OXA-1 USING CRTSPR7CAS RIBONUCLEOPREINE COMPLEXES USING A MODEL MATRIX

The pre-amplified material obtained by the method described in Example 2 was used as a template for detection of the antibiotic resistance gene bla-OXA-1 using CRISPR-Cas ribonucleoprotein complexes obtained by the method described in Example 3.

To detect the antibiotic resistance gene bla-OXA-1 using CRISPR-Cas ribonucleoprotein complexes, a reaction mixture was prepared containing the following components:

• 10×buffer (100 mM TrisHCl pH 8.0, 1 M NaC1);

• 50 mM MgCl ₂ (final concentration in the reaction mixture 10 mM);

• 250 ng ribonucleoprotein complex (LbCpfl from Lachnospiraceae and guide RNAs crRNA bla-OXA-1 No. 63, crRNA bla-OXA-1 No. 185 and crRNA bla-OXA-1 No. 221);

• 10 pmol fluorescent probe (6FAM-TTATT-BHQ1);

• Target (pre-amplified fragment of the antibiotic resistance gene bla-OXA-1);

• water mQ.

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

30-60 cycles:

1. 37°С - 35 sec,

2. 37°C - 25 sec, fluorescence imaging.

First of all, experiments were conducted to detect single copies of the antibiotic resistance gene bla-OXA-1 using CRISPR-Cas ribonucleoprotein complexes formed on the basis of LbCpfl from Lachnospiraceae , using as a target a model matrix - plasmid DNA pGEM-T-bla-OXA-1, containing in its composition a fragment of the antibiotic resistance gene bla-OXA-1 of 321 bp.

CRISPR-Cas ribonucleoprotein complexes have been shown to be able to detect single copies of the bla-OXA-1 antibiotic resistance gene. Representative results of the assay are shown in real-time fluorescence profiles of a pre-amplified bla-OXA-1 antibiotic resistance gene fragment treated with ribonucleoprotein complexes containing the guide RNAs crRNA bla-OXA-1#63, crRNA bla-OXA-1#185, and crRNA bla-OXA-1#221 and the LbCpfl protein in Fig. 2, Fig. 3, and Fig. 4, respectively.

It should be noted that CRISPR-Cas 12 complexes detect the bla-OXA-1 gene with varying efficiency. Thus, for crRNA bla-OXA-1 #63 and crRNA bla-OXA-1 #221, on average, already at the 5th analysis cycle (5 minutes), the signal value obtained during the detection of single copies (1.25 copies/reaction) of the antibiotic resistance gene bla-OXA-1 exceeded the “noise” value (non-specific fluorescence of the control sample not containing the target) by at least five times, and by the 8th cycle (8 minutes of analysis) - by 10 or more times. Whereas for crRNA bla-OXA-1 No. 185, the signal value obtained during detection of single copies (1.25 copies/reaction) of the antibiotic resistance gene bla-OXA-1 exceeded the “noise” value (non-specific fluorescence of the control sample not containing the target) by 5 times by the 23rd cycle (23 minutes of analysis) and by 10 or more times by the 32nd cycle (32 minutes).

In the course of the work, the efficiency of detection of the antibiotic resistance gene bla-OXA-1 contained in the model matrix was assessed using various guide RNAs. It was shown that the CRISPR-Cas ribonucleoprotein complexes formed on the basis of LbCpfl from Lachnospiraceae and guide RNAs detect the antibiotic resistance gene bla-OXA-1 with varying efficiency, and they can be arranged in the following order by decreasing activity: crRNA bla-OXA-1 #63 ≈ crRNA bla-OXA-1 #221>crRNA bla-OXA-1 #185 (Fig. 5).

EXAMPLE 5: DETECTION OF THE ANTIBIOTIC RESISTANCE GENE BLA-OXA-1 USING CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES IN A LIMITED PANEL OF CLINICAL SAMPLES

The developed guide RNAs were tested on a limited panel of clinical samples (10 pcs.) containing the antibiotic resistance gene bla-OXA-1 (previously confirmed by next-generation sequencing).

To detect the antibiotic resistance gene bla-OXA-1 using CRISPR-Cas ribonucleoprotein complexes, preliminary amplification of the antibiotic resistance gene fragment bla-OXA-1 was performed. To perform preliminary amplification, DNA preparations were isolated from clinical samples of 10 patients using the commercially available RIBO-prep kit (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) according to the manufacturer's instructions.

The product encoding the fragment of the antibiotic resistance gene bla-OXA-1 was obtained by PCR using specific oligonucleotides OXA-l_for_2 and OXA-1_rev_1 (GenTerra, Russia) and visualized by electrophoresis in agarose gel (Fig. 6).

The material obtained in this way was used as a matrix for detection of the antibiotic resistance gene bla-OXA-1 using CRISPR-Cas ribonucleoprotein complexes obtained by the method described in Example 3.

Detection of the antibiotic resistance gene bla-OXA-1 using CRISPR-Cas ribonucleoprotein complexes was performed as described in Example 4.

The analysis showed that CRISPR-Cas ribonucleoprotein complexes have the ability to detect the antibiotic resistance gene bla-OXA-1 in DNA preparations isolated from clinical samples. At the same time, on average, already at the 7th cycle (7 minutes) of the analysis, the signal value exceeded the "noise" value (non-specific fluorescence of the control sample not containing the target) by more than 5 times, and by the 8th cycle (8 minutes) of the analysis - by more than 10 times (Table 4).

Typical assay results are shown in the examples of end-point fluorescence values (60 assay cycles, 60 minutes) for a pre-amplified fragment of the antibiotic resistance gene bla-OXA-1 (10 independent clinical samples) treated with ribonucleoprotein complexes containing guide RNAs crRNA bla-OXA-1 #63, crRNA bla-OXA-1 #185 and crRNA bla-OXA-1 #221 and the LbCpfl protein in Fig. 7.

The efficiency of detection of the antibiotic resistance gene bla-OXA-1 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 , estimated by the ratio of signal to noise values during detection, can be presented in the following descending order: crRNA bla-OXA-1 #221 > crRNA bla-OXA-1 #63 > crRNA bla-OXA-1 #185 (Table 4).

Thus, the developed guide RNAs allow ultra-sensitive detection of single copies of the antibiotic resistance gene bla-OXA-1, and are capable of detecting it in DNA preparations isolated from clinical samples after preliminary amplification as part of CRISPR-Cas ribonucleoprotein complexes.

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Claims (9)

1. A method for detecting the antibiotic resistance gene bla-OXA-1, comprising: i) performing preliminary amplification of the sample material from the patient, presumably containing the antibiotic resistance gene bla-OXA-1, using one or more specific oligonucleotides selected from SEQ ID NO: 4-5, in order to obtain a target - a pre-amplified fragment of the antibiotic resistance gene bla-OXA-1, ii) preparing a detection reaction mixture comprising a 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: 1-3; a fluorescent probe and a detection buffer, iii) carrying out 30-60 detection cycles in the reaction mixture obtained in step (ii) in a thermocycler, with the discovery of the antibiotic resistance gene bla-OXA-1. 2. The method according to claim 1, wherein the pre-amplified fragment of the antibiotic resistance gene bla-OXA-1 is represented by a 262 bp fragment of the antibiotic resistance gene bla-OXA-1. 3. The method according to claim 1 or 2, wherein the method ensures the detection of the antibiotic resistance gene bla-OXA-1 in ultra-low concentrations, down to single copies of DNA. 4. A specific oligonucleotide selected from SEQ ID NO: 4-5 for use in the method according to claim 1, 2 or 3 for preliminary amplification of a highly conserved fragment of the antibiotic resistance gene bla-OXA-1, which is recognized by the ribonucleoprotein complex. 5. A kit for use in the method according to claim 1, 2 or 3, comprising 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: 1-3; a fluorescent probe, a detection buffer and instructions for use.