RU2685188C2 - Dna-chip for identifying genetic determinant of antibiotic resistance of infectious agents leading to disturbed human reproductive functions, kit of oligonucleotides for immobilization on dna-chip - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to biotechnology, biochemistry, molecular biology, microbiology and medicine. Disclosed is a DNA chip and a kit for identifying genetic determinants of resistance of microorganisms – infectious agents, leading to disturbed human reproductive functions: Treponema pallidum, Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Gardnerella vaginalis, Ureaplasma parvum, Ureaplasma urealiticum, Atopobium vaginae, Enterococcus faecalis, Trichomonas vaginalis, Escherichia coli, Mobiluncus mulieris, Streptococcus anginosus, Fusobacterium nucleatum, Staphylococcus epidermidis, Bacteroides fragilis, to antimicrobial preparations containing penicillin antibiotics, cephalosporins, tetracyclines, fluoroquinolones, macrolides, nitroimidazoles, spectinomycin.EFFECT: invention can be used in medicine for effective personified therapy of diseases, clinical laboratory diagnostics, epidemiology.2 cl, 22 dwg, 4 tbl, 2 ex

Description

The technical field to which the invention relates.

The invention relates to molecular biology, microbiology and medicine, and provides a DNA chip for identifying genetic determinants of antibiotic resistance of infectious pathogens that lead to impaired human reproductive functions (IPNRF): Treponema pallidum, Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma publ, mygra mama, myla, 100% Ureaplasma, Ureaplasma, Atopobium vaginae porins, tetracyclines, fluoroquinolones, macrolides, nitroimidazoles, spectinomycin, as well as a set of oligonucleotides for immobilization on a DNA chip.

The level of technology

According to the International Classification of Diseases (ICD-10, http://www.medicusamicus.com/index.php?action=mkb), infections that cause diseases of the urogenital tract and lead to violations of human reproductive functions include syphilis, gonococcal infection, urogenital trichomoniasis, chlamydia infection, etc. The pathological conditions of the genitourinary system are often polymicrobial in nature, represented not only by obligate pathogens (Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Mycoplasma genitalium), but also by conditionally pathogenic infectious agents, but also by means of agents who are infected with a pattern, they can also be used by pattern agents, they can also be used by pattern agents, they can also be used by pattern agents. es, Fusobacterium, Ureaplasma, Streptococci, Staphylococci, Enterococci, E. coli, Gardnerella vaginalis, etc.). A serious problem throughout the world is the growth of drug resistance of pathogens, which causes considerable difficulties in the choice of methods for treating patients. A WHO report on “Antimicrobial Resistance: A Global Surveillance Report” (April 2014) presents a high risk of increased resistance to antimicrobials for a number of infectious agents, and among the infections of greatest concern, along with tuberculosis, malaria, staphylococcal infections, N. gonorrhoeae (the appearance of strains with reduced sensitivity to third-generation cephalosporins) and E. coli (resistance to fluoroquinolones and cephalosporins) were isolated. Thus, to determine the etiology of the infectious-inflammatory process of the urogenital system and subsequent successful therapy, it is necessary not only to identify infectious agents in clinical specimens, including the simultaneous identification of a large number of pathogens, but also to analyze the sensitivity / resistance of infectious agents to the main antimicrobial agents. Due to the extraordinary diversity of resistance mechanisms of the causative agents of the CPRP, the methods being developed should cover the maximum possible range of molecular parameters associated with resistance (Shaskolskiy B, Dementieva E, Leinsoo A, Runina A, Vorobyev D, Plakhova X, Kubanov A, Deryabin D, Gryadunov D. 2016 Drug resistance mechanisms in bacteria causing sexually transmitted diseases and associated with vaginosis (Frontiers in Microbology. 7: 747).

The main methods of laboratory diagnosis of diseases are microbiological (microscopic and cultural research) and molecular biological (polymerase chain reaction (PCR), NASBA (Nucleic Acids Sequence-Based Amplification) methods of research, determination of the nucleotide sequence of microorganism sites, in particular, detection of mutations leading to resistance to drugs. Microscopic examination is the most accessible method, but its sensitivity in identifying a number of infectious agents It does not exceed 40-60% due to the subjective assessment of the results of the analysis. A cultural study has high specificity and sensitivity (96-100%), however it is expensive and time-consuming, which limits its widespread use. Cultivation of a number of microorganisms (M genitalium, Mobiluncus, Peptostreptococcus, Prevotella, Bacteroides, Fusobacterium, etc.) is impossible in the conditions of laboratories of medical institutions due to the high demands on nutrient media and the duration of their cultivation (up to 5 months), and T. pallidum is not cultivated in vitro.

Identification of microorganisms is also performed using the method of mass spectrometry, which allows for the analysis not only of pure cultures, but also of biological material. The disadvantages of microbiological mass spectrometry include the imperfection of identification of microorganisms in mixed cultures and the lack of standard criteria for evaluating antibiotic resistance (Seng R., Drancourt M. Gouriet F., La Scola V., Fournier PE, Rolain JM, Raoult D. Ongoing revolution bacteriology : matrix identification, laser identification, ionization time-of-flight mass spectrometry, Clin Infect Dis, 2009 v. 49, p. 543-551.). The high cost of equipment and personnel qualifications allow using this approach only in large centralized bacteriological laboratories.

, Франция), ВАСТЕС (Becton Dickinson, США), Sensititre TREK Diagnostic Systems (Thermo Scientific, США). С использованием данных систем можно получить количественную информацию об уровне устойчивости микроорганизмов к различным препаратам, однако время проведения анализа для получения данных о профиле резистентности составляет от 7 суток до нескольких недель.To determine the phenotypic resistance of microorganisms to antimicrobial drugs using methods of serial dilution on agar and disco-diffusion method. The “gold standard” for microbiological diagnosis of antibiotic resistance is the serial dilution method. Automated systems for cultivating microorganisms and determining their drug sensitivity, such as VITEK (

, France), WASTES, Becton Dickinson, USA), Sensititre TREK Diagnostic Systems (Thermo Scientific, USA). Using these systems, it is possible to obtain quantitative information about the level of resistance of microorganisms to various drugs, however, the analysis time for obtaining data on the resistance profile ranges from 7 days to several weeks.

In recent years, the basis of laboratory diagnostics in many countries of the world consists of nucleic acid amplification methods. The advantages of PCR methods include speed, the ability to automate and simultaneously conduct a large amount of research, the ability to conduct research directly with biological material, bypassing the stage of cultivation of the microorganism. However, various inhibitory factors may affect the sensitivity of the study, as a result of which false-negative results are possible. The disadvantages of this method can also be attributed to the greater demands on the purity of the reagents used.

In the literature there are works devoted to the simultaneous identification of a number of pathogens of IPNRF based on PCR amplification followed by the detection of products by hybridization, gel electrophoresis, membrane blotting, etc. The method of carrying out PCR for the simultaneous detection of 7 pathogens is described: N. gonorrhoeae, C. trachomatis , T. vaginalis, M. genitalium, T. pallidum, herpes viruses (type 1 and 2) (Muvunyi C.M., Dhont N., Verhelst R., et al. Evaluation of a new multiplex polymerase chain reaction STD finder Sexual disease, pathogens, Diagnostic Microbiol. Disfect. 2011, v. 71 (1), p. 29-37. The method of multiplex PCR and linear blot for simultaneous identification of 14 microorganisms, including T. vaginalis, S. pneumoniae, N. gonorrhoeae, C. trachomatis, U. parvum, U. urealyticum, G. vaginalis, H. influenzae, HSV-1, HSV-2, N Meningitidis, M. hominis, M. genitalium, adenoviruses Detection was performed using a chemiluminescent reaction. Two specific pairs of probes were used for each organism (McKechnie ML, Kong F., Gilbert GL) using multiplex PCR-based reverse-line blot assays. Methods Mol. Biol., 2013; v. 943, p. 229-245).

A test system based on multiplex PCR and reverse linear blot was developed to identify 22 factors of E. coli virulence causing the development of non-gonococcal urethritis (Kudinha T., Kong F., Johnson JR, Andrew SD, Anderson P., Gilbert, GL Multiplex PCR-based virulence genes in uropathogenic Escherichia coli, reverse line blot assay (Applied and Environmental Microbiology, 2012, v. 78, p. 1198-1202).

A PCR method for the simultaneous screening of 13 agents associated with bacterial vaginosis is described: G. vaginalis, M. curtisii, M. mulieris, B. fragilis, M. hominis, A. vaginae, U. urealyticum, Megasphaera (type I), Clostridia , Sneathia sanguinegens, M. genitalium (Malaguti N., Bahls LD, Uchimura NS, Gimenes F., Consolaro ME, 2014. Epub 2015 May 20, doi: 10.1155 / 2015/645853).

A number of commercial test systems for the simultaneous diagnosis of two or more IPNRF pathogens have been developed, for example, Amplisens kits (InterLabService, Russia), Bio-Rad CT / NG / MG assay (USA), Abbott RealTime CT / NG and APTIMA Combo 2 Assay (Abbott, USA), Roche Cobas 4800 CT / NG assay (Roche, USA), Anyplex II STI-7 assay (TOTEM technology, USA). STI multiplex array ('Randox', Ireland), BD Probetec ™ ET - CT / GC (Becton Dickinson, USA). The detection sensitivity is 500 cells / ml. None of the existing commercial test systems allows the identification of genetic determinants of antimicrobial resistance at the same time as identification.

In all the above works, the analysis of drug resistance of pathogens was not carried out.

Published work on the use of PCR methods for the analysis of mutations leading to drug resistance of one pathogen, for example, N. gonorrhoeae (Dona V., Kasraian S., Lupo A., Guilarte YN, Hauser C., Furrer H., Unemo M. , Low N., Endimiani A. A Multiplex Real-Time PCR for Neisseria gonorrhoeae (J. Clin. Microbiol, 2016. May 25. pii: JCM.03354-15).

To carry out a multiparameter analysis of nucleic acids, a technology called CMAR is proposed, in which the analysis is carried out using a suspension of fluorescently labeled microspheres, on the surface of which oligonucleotide probes are located. The use of microspheres with detection of Luminex for the analysis of 18 microorganisms that cause urogenital infections is described: C. trachomatis, herpes viruses (type 1 and 2), T. pallidum, T. vaginalis, N. gonorrhoeae, M. genitalium, M. hominis, M. pneumonia, M. spermatophilum, Ureaplasma urealyticum, U. parvum, A. vaginalis, G. vaginalis, 3 species of Candida and Lactobacillus species (Schmitt M., Depuydt C., Stalpaert M., Pawlita, M. Bead-based multiplex sexually identified infection profiling. Journal of Infection, 2014, v. 69, p. 123-133). Balashov S. and co-authors (Balashov S., Mordechai E., Adelson ME, Gygax SE Multiplex bead suspension for screening Neisseria gonorrhoeae antibiotic genetic determinants in noncultured clinical samples. J. Mol. Diagn., 2013, v. 15, p. 116 -129) proposed a combination of PCR and multiplex profiling on MicroPlex-xTAG microspheres (Luminex Corp., USA) for the simultaneous detection of 29 genomic mutations and 2 plasmid N.gonorrhoeae genes that serve as genetic markers of N.gonorrhoeae resistance to 6 antimicrobial drugs (penicillin 6 antimicrobial drugs (penicillitis). , ciprofloxacin, cefixime, tetracycline, azithromycin and spectinomycin). The test system is designed to work only with N. gonorrhoeae and is not applicable for the identification of other pathogens IPNRF.

Methods of targeted sequencing using modern systems, for example, Ion PGM sequencer (Life technologies, USA), MiSeqDx Instrument (Illumina, USA) make it possible to identify a sample and simultaneously determine the genetic determinants of antibiotic resistance, however, the sensitivity of these methods is insufficient for analyzing clinical material. The methods are still distinguished by high cost, and therefore they are not currently being widely used in routine laboratory diagnostics of the causative agents of IPNRF.

Modern methods of multiparameter analysis of genomic targets also include manipulations (hybridization, amplification) with nucleic acids on DNA microarrays (microchips) - arrays of oligonucleotide probes fixed on the solid phase that can specifically bind to the analyzed genome fragments. Several variants of DNA microchips have been patented for the simultaneous detection of several pathogens of IPNRF. The State Scientific Center for Dermatovenerology and Cosmetology of the Ministry of Health of the Russian Federation has developed a DNA chip for the simultaneous detection of 28 pathogenic, non-pathogenic and conditionally pathogenic microorganisms of the human genitourinary sphere, which is detected after multiplex PCR (the DNA chip for the integrated diagnosis of infections transmitted sexually, "RF patent for utility model 117431, priority from 05.11.2009; Likhareva V.V., Frigo N.V., Rakhmatulina MR, Nurutdinova OS, Shatalova, Yu.A. chip for integrated diagnostician iki IPPP. Vestn. dermatol. venerol., 2009, t. 4, p. 49-57). Biochip for the simultaneous species identification of several infectious agents is patented by Molecular Medical Technology CJSC (RF patent No. 2348695, priority date 05/23/2006, Differentiating and specific oligonucleotides for identifying the DNA sequences of infectious agents in biological materials, the method of species identification of infectious agents, biochip and a set for implementing this method ").

There is a patent by Korean researchers for a DNA chip intended for the diagnosis of urinary tract infections (United States patent application number 201340336082). A DNA microchip for simultaneous anazis of 17 pathogens of sexually transmitted diseases is described: N. gonorrhoeae, C. trachomatis, M. genitalium, M. hominis, Ureaplasma, herpes viruses 1 and 2 types, papillomaviruses of various types (Sao V., Wang S ., Tian Z., P. P. Hu, Feng L., Wang L. DNA microarray characterization of pathogens associated with sexually transmitted diseases, PLoS One., 2015, v. 10 (7): e0133927). The chip contained 34 probes including fragments of 16S RNA. The above DNA chips are intended only for the detection of infectious agents, but not for identifying the determinants of drug resistance.

Published oligonucleotide microchip for the detection of point mutations in the gyrA and parC genes of N. gonorrhoeae, leading to drug resistance (Zhou W., Du W., et al. Detection of gyrA and parC mutations associated with ciprofloxacin resistance in Neisseria gonorrhoeae by oligonudele technology. J. Clin. Microbiol., 2004, v. 42 (12), p. 5819-5824). The microchip allows detection of 15 determinants leading to resistance exclusively to ciprofloxacin, in particular, mutations of residues S91 and D95 of the GyrA protein, residues E91 and S87 of the ParC protein.

Thus, the currently known methods of molecular multiplex diagnosis of pathogens IPNRF solve either the problem of identifying microorganisms or determining markers of resistance to antimicrobial agents, as a rule, only for one type of microorganism. There is a need for the development of molecular genetic tool for the simultaneous identification of some of the most common pathogens IPNRF and molecular determinants of resistance to a broad spectrum topical antimicrobial agents such as antibiotics, penicillins, cephalosporins, tetracyclines, fluoroquinolones, macrolides, nitroimidazoles, spectinomycin. The use of this method will allow for personalized therapy of patients, based on the rational prescription of chemotherapy drugs, to significantly reduce the number of complications and failures in the treatment of inflammatory diseases of the urogenital tract caused by microorganisms resistant to antimicrobial drugs.

DISCLOSURE OF INVENTION

The invention proposes a tool for the simultaneous identification of a number of the most common pathogens of IPNRF, as well as molecular determinants of their resistance to a wide range of topical antimicrobial agents in the form of a DNA chip with immobilized oligonucleotide probes. Invented djs vaginalis, Escherichia coli, Mobiluncus mulieris, Streptococcus anginosus, Fusobacterium nucleatum, Staphylococcus epidermidis, Bacteroides fragilis to antimicrobial drugs, including penicillin antibiotics, cefalosporins, tetracyclines, aph, and, in a pattern, non-microscopes, anti-microbials, penicillin antibiotics, cefalosporins, tetracyclines, tetracyclines, antibiotics. ching, which makes it possible to establish an accurate diagnosis based on polymicrobial nature of the disease and prescribe effective personalized therapy.

In its first aspect, this invention provides a DNA chip, which is a matrix of elements placed on a glass or plastic substrate, the matrix comprising:

- elements with immobilized oligonucleotides with nucleotide sequences of SEQ ID NO: 1-33, corresponding to 16S rRNA of T. pallidum, N. gonorrhoeae, C. trachomatis, M. genitalium, M. hominis, G. vaginalis, U. parvum, U. urealyticum , A. vaginae, E. faecalis, T. vaginalis, E. coli, M. mulieris, S. anginosus, F. nucleatum, S. epidermidis, B. fragilis to establish the belonging of the analyzed DNA to the above-mentioned types of microorganisms that are infectious leading to impaired human reproductive functions;

- elements with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 34-42, corresponding to the sequences of the gyrA gene of wild-type N. gonorrhoeae isolates, or sequences of mutant versions of the gyrA gene, resulting in S91F, S91T, D95N, D95G mutant variants of the isolates, which are found in isolates. Fluoroquinolone-resistant gonorrhoeae;

- Elements with immobilized oligonucleotides with nucleotide sequences of SEQ ID NO: 43-55, corresponding to the sequences of the gyrA gene of wild-type E. coli isolates, or sequences of mutant versions of the gyrA gene, resulting in amino acid substitutions of S83L, S83W, S83A, D87N, D87G, D87Y, the meetings in E. coli isolates resistant to fluoroquinolones;

- elements with immobilized oligonucleotides with the nucleotide sequences SEQ ID NO: 56-58, corresponding to sequences gyrA gene S. epidermidis isolates of wild-type or mutant sequences embodiment gyrA gene leading to amino acid substitutions S83T, S83F, occurring in S. epidermidis isolates, resistant to fluoroquinolones;

- elements with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 59-63, corresponding to the sequences of the gyrA gene of wild-type F. nucleatum isolates, or sequences of mutant versions of the gyrA gene, leading to amino acid substitutions S83L, S83T, G87R, occurring in F. nucleatum isolates resistant to fluoroquinolones;

- elements with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 64-67, corresponding to the sequences of the gyrA gene of wild-type E. faecalis isolates, or sequences of mutant versions of the gyrA gene, leading to amino acid substitutions S83T, S83I, S83R, found in E. feeisis isolates resistant to fluoroquinolones;

- elements with immobilized oligonucleotides with nucleotide sequences of SEQ ID 68-73, corresponding to the sequences of the gyrA gene of wild-type S. anginosus isolates, or sequences of mutant versions of the gyrA gene, resulting in amino acid substitutions S83T, S83F, which are found in S. anginosus resistant sequences of resistant antibodies to the S83T, S83F, which are resistant to S. anginosus, resistant antibodies to the S83T, S83F isolates found in resistant S. ;

- elements with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 74-82, corresponding to the sequences of the parC gene of wild-type N. gonorrhoeae isolates, or sequences of mutant parC gene variants leading to amino acid substitutions S87N, S87R, E91Q, E91G, E91K, E91A found in N. gonorrhoeae isolates resistant to fluoroquinolones;

- elements with immobilized oligonucleotides with nucleotide sequences of SEQ ID NO: 83-90, corresponding to the sequences of the parC gene of M. wild type genitalium isolates, or sequences of mutant variants of the parC gene, leading to amino acid substitutions S83N, S83I, S83R, D87N, D87Y, D87V, found in M. genitalium isolates resistant to fluoroquinolones;

- elements with immobilized oligonucleotides with nucleotide sequences of SEQ ID NO: 91-95, corresponding to the sequences of the parC gene of wild type h. hash isolates, or sequences of mutant variants of the parC gene, leading to amino acid substitutions S83I, S83N, E87K, occurring in M. hominis isolates resistant to fluoroquinolones;

- elements with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 96-99, corresponding to the sequences of the parC gene of wild-type U. ualyutumum isolates, or sequences of mutant variants of the parC gene, leading to amino acid substitutions D82N, S83L, found in U. urealyticum, isolates, which are found in isolates of U. urealyticum, which he has found in amino acids D82N, S83L, which are found in U. urealyticum, isolates, which are found in isolates of U. urealyticum, which are found in isolates of U. to fluoroquinolones;

- elements with immobilized oligonucleotides with the nucleotide sequences SEQ ID NO: 100-103, the relevant gene sequences ntr4 _tv and ntr6 _tv T. vaginalis isolates of wild-type or mutant sequences of variants with nucleotide substitutions C213G (Y71STOP) gene ntr4 _tv, A238T ( K80STOP) in the ntr6 _tv gene found in T. vaginalis isolates resistant to nitroimidazoles;

- elements with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 104-109, corresponding to the sequences of the genes nimB, nimC, nimD, nimE, nimF, nimG, the presence of which leads to the resistance of the microorganism (s) to nitroimidazoles;

- an element with immobilized oligonucleotides with the nucleotide sequence of SEQ ID NO: 110, corresponding to the sequence of the _SHV-12 bla _LEN-17 plasmid genes, the presence of which leads to resistance of the microorganism (s) to penicillin antibiotics and cephalosporins;

- an element with immobilized oligonucleotides with the nucleotide sequence of SEQ ID NO: 111, corresponding to the sequence of the plasmid gene _SHV-97 bla, the presence of which leads to the resistance of the microorganism (s) to penicillin antibiotics;

- elements with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 112-117, corresponding to the sequences of the plasmid genes bla _TEM and bla _SHV , which result in resistance of the microorganism (s) to penicillin antibiotics, or sequences of the mutant variant of the gene bla _TEM with amino acid substitutions M182T and G238S, the simultaneous presence of which leads to the resistance of the microorganism (s) to penicillin antibiotics and cephalosporins;

- elements with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 118-119, corresponding to the sequences of the penA gene of wild-type N. gonorrhoeae isolates, or sequences of the mutant variant of the penA gene, leading to an insertion of aspartic acid (D) at the position 345 found in isolates N Penicillin-resistant antibiotic-resistant gonorrhoeae;

- elements with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 120-123, the corresponding sequences of the ponA gene of wild-type N. gonorrhoeae isolates, or the sequences of the mutant variant of the ponA gene with the amino acid substitution L421P found in N. gonorrhoea-resistant, resistant, resistant amino acid sequences of the gene of N. gonorrhoeae, an amino acid substitution of the N. ;

- elements with immobilized oligonucleotides with the nucleotide sequences SEQ ID NO: 124-126, the relevant gene sequences rpsJ N. gonorrhoeae isolates of wild-type or mutant sequences embodiment rpsJ c gene amino acid substitution V57M, V57L, occurring isolates from N. gonorrhoeae, tetracycline resistant ;

- elements with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 127-128, corresponding to the sequences of the tetM genes, whose presence in E. faecalis, E. coli, M. genitalium, M. hominis, N. gonorrhoeae, G. vaginalis leads to resistance to tetracyclines;

- an element with immobilized oligonucleotides with the nucleotide sequence of SEQ ID NO: 129, corresponding to the tetO gene sequence, whose presence in E. faecalis, S. epidermidis isolates leads to tetracycline resistance;

- elements with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 130-139, corresponding to the sequences of 23S RNA isolates from U. urealyticum, U. hominis, S. anginosus, F. nucleatum, E. faecali, T. pallidum, E. coli, N gonorrhoeae, C. trachomatis, M. genitalium, M. hominis, S. epidermidis, wild-type B. fragilis, or sequences of mutant variants with substitutions G2057A, A2058G, A2058C, A2059G, found in isolates of the listed pathogens resistant to macrolides;

- elements with immobilized oligonucleotides with the nucleotide sequences of SEQ ID NO: 140-141, corresponding to the mefA gene sequence, the presence of which leads to the resistance of S. anginosus, N. gonorrhoeae, S. epidermidis, B. fragilis to macrolides;

- elements with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 142-150, corresponding to the sequences of 16S RNA isolates of wild-type N. gonorrhoeae, or sequences of mutant variants with substitutions of С1192Т, G1064C, found in isolates of N. gonorrhoeae, resistant to spectin-infected mycotic cells, G1064C, resistant to spectin-resistant specimens, resistant to spectin.

- elements with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 148, 151-155, corresponding to the sequences of 16S RNA isolates of C. trachomatis, E. coli, F. nucleatum, A. vaginae, T. pallidum, U. urealyticum, U. hominis , S. anginosus, S. epidermidis, M. genitalium, M. hominis, M. mulieris, B. fragilis of the wild type, or sequences of mutant variants with G1058C, G1064C substitutions found in isolates of the listed pathogens resistant to tetracycline;

- Elements with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 156-161, corresponding to sequences of the wild-type mtrR pump efflux gene, or sequences of mutant variants of the mtrR promoter region, leading to deletion of adenine A at -35 or insertions of thymidine T or TT position -10, found in isolates of pathogens resistant to penicillin antibiotics, tetracyclines, macrolides, cephalosporins;

- Elements with immobilized oligonucleotides with the nucleotide sequences of SEQ ID NO: 162-163, corresponding to the gene sequence of the porin protein porB of wild-type N. gonorrhoeae isolates, or the sequence of the mutant variant of the porB gene, resulting in the G120K amino acid substitution found in N. gonorrhoee isolates, which plaques, the recipient gene, which results in an amino acid substitution G120K found in the isolates of N. gonorrhoee, which are used by the registrant, the gene for the gene of the mutant variant of the porB gene. to penicillin antibiotics, cephalosporins, tetracyclines.

Another aspect of the present invention is a set of oligonucleotides used to obtain a DNA chip to identify the genetic determinants of resistance of microorganisms that are pathogens of AIPNRF: T. pallidum, N. gonorrhoeae, C. trachomatis, M. genitalium, M. hominis, G. vaginalis, U . parvum, U. urealyticum, A. vaginae, E. faecalis, T. vaginalis, E. coli, M. mulieris, S. anginosus, F. nucleatum, S. epidermidis, B. fragilis, to antimicrobial drugs, including penicillin antibiotics series, cephalosporins, tetracyclines, fluoroquinolones, macrolides, nitroimidazoles, spectinomycin, and the oligonucleotides have ti SEQ ID NO: 1-163.

The list of sequences of oligonucleotides used to manufacture the DNA chip, and their SEQ ID are shown in Table 1.

List of figures

Figure 1. Layout of the elements of the DNA chip for identification of genetic determinants of resistance of pathogens IPRNF T. pallidum, N. gonorrhoeae, C. trachomatis, M. genitalium, M. hominis, G. vaginalis, U. parvum, U. urealyticum, A. vaginae, E. faecalis, T. vaginalis, E. coli, M. mulieris, S. anginosus, F. nucleatum, S. epidermidis, B. fragilis to antimicrobial drugs, including penicillin antibiotics, cephalosporins, tetracyclines, fluoroquinolones, macrolides, nitroimidazoles, spectinomycin. Elements of a DNA chip are presented as circles, the inscriptions inside which indicate the purpose of the elements: analysis of the belonging of the analyzed DNA to the species of the causative agent of IPNRF, analysis of variable amino acids / nucleotides for compliance with the wild type or mutant variant of the isolate of the causative agent of IPNRF; identification of the resistance marker gene (gene name).

M - elements containing a fluorescent marker;

O - elements that do not contain immobilized oligonucleotides, which are used to calculate the background signal.

The arrangement of the oligonucleotide probes immobilized on the DNA chip in accordance with the SEQ ID is given in Table 2.

Figures 2-22. Hybridization patterns of DNA chips and the results of their interpretation in the analysis of DNA samples of isolates of pathogens IPNRF, different profiles of antibiotic resistance.

The implementation of the invention

The aim of the invention was to design a tool for determining the molecular determinants of resistance of a number of the most common causative agents of AIPNRF to a wide range of topical antimicrobial agents. This invention proposes a DNA chip - matrix of elements with immobilized oligonucleotide probes for identifying the genetic determinants of resistance of T. pallidum, N. gonorrhoeae, C. trachomatis, M. genitalium, M. hominis, G. vaginalis, U. parvum, U. urealyticum , A. vaginae, E. faecalis, T. vaginalis, E. coli, M. mulieris, S. anginosus, F. nucleatum, S. epidermidis, B. fragilis, to antimicrobial drugs, including penicillin antibiotics, cephalosporins, tetracyclines, fluoroquinolones, macrolides, nitroimidazoles, spectinomycin, as well as a set of oligonucleotides used to obtain a DNA chip.

A DNA chip is a glass or plastic substrate on which oligonucleotide probes are attached. Oligonucleotides can be adsorbed on a substrate, fixed (immobilized) on a substrate through the formation of covalent bonds, through hydrophobic interactions, complexation, etc. A DNA chip can be obtained by applying aqueous solutions of oligonucleotides directly to the substrate to obtain a variant of the so-called “planar” (two-dimensional) chip (for example, according to Matson RS Microarray Methods and Protocols. 2009. CRC Press, ISBN 9781420046656), or be an array of three-dimensional hemispherical hydrogel cells containing covalently immobilized oligonucleotides. A three-dimensional DNA chip can be manufactured, for example, by the method of copolymerization immobilization (RF patent №2216547, patent EP 1437368; Rubina AY, Pan'kov SV et al., Hydrogel drop microchips with immobilized DNA). Anal. Biochem., 2004, v. 325, p. 92-106). One of the ways to obtain a DNA chip is shown in Example 1.

The layout of the oligonucleotides on the DNA chip is shown in FIG. 1. The biochip contains 168 elements, including 163 cells with immobilized oligonucleotides, 1 cell that does not contain probes, to calculate the background signal (the cell with the “O” index), and 4 cells, containing the immobilized marker (cells with the “M” index). The cells with the marker are used for the correct positioning of the DNA chip during the registration of the hybridization pattern and its processing by the biochip analyzer software (scanner) when automatically obtaining results.

The DNA chip allows to establish the belonging of the analyzed DNA to the species of microorganisms that are infectious agents, including T. pallidum, N. gonorrhoeae, C. trachomatis, M. genitalium, M. hominis, G. vaginalis, U. parvum, U. urealyticum, A vaginae, E. faecalis, T. vaginalis, E. coli, M. mulieris, S. anginosus, F. nucleatum, S. epidermidis, B. fragilis, based on species-specific polymorphism in the 16S rRNA genes. For this purpose, elements with immobilized oligonucleotide probes with sequences having SEQ ID NO: 1-33 are used.

The selection of the analyzed genetic determinants of resistance was carried out taking into account the analysis of modern literature, so as to cover the widest possible range of relevant clinically significant mechanisms for the formation of antimicrobial pathogens of AIPNRF (Shaskolskiy B, Dementieva E, Leinsoo A, Runina A, Vorobyev D, Plakhova X, Kubanov A, Deryabin D, Gryadunov D. 2016. Frontiers in Microbology (7: 747).

At the same time, the DNA chip allows you to determine:

- mutations in the gene of gyrA N. gonorrhoeae S91F, S91T, D95N, D95G, found in the isolates of N. gonorrhoeae resistant to fluoroquinolones. Elements are used with immobilized oligonucleotide probes with sequences having SEQ ID NO: 34-42;

- mutations in the gyrA gene of E. coli S83L, S83W, S83A, D87N, D87G, D87Y, found in E. coli isolates resistant to fluoroquinolones. Elements with immobilized oligonucleotide probes with sequences having SEQ ID NOs: 43-55;

- mutations in the gyrA gene of S. epidermidis S83T, S83F, which are found in S. fluxinolone-resistant S. epidermidis isolates. Elements with immobilized oligonucleotide probes with sequences having SEQ ID NOs: 56-58;

- mutations in the gyrA gene of F. nucleatum S83L, S83T, G87R, found in F. fluorequinolone-resistant isolates of F. nucleatum. Elements with immobilized oligonucleotide probes with sequences having SEQ ID NO: 59-63;

- mutations in the gyrA gene of E. faecalis S83T, S83I, S83R, found in E. faecalis isolates resistant to fluoroquinolones. Elements with immobilized oligonucleotide probes with sequences having SEQ ID NO: 64-67;

- mutations in the gyrA gene of S. anginosus S83T, S83F, found in S. fluoquinolone-resistant S. anginosus isolates. Elements are used with immobilized oligonucleotide probes with sequences having SEQ ID NO: 68-73;

- mutations in the gene of parC N. gonorrhoeae S87N, S87R, E91Q, E91G, E91K, E91A, found in N. flutequinolone-resistant N. gonorrhoeae isolates. Elements are used with immobilized oligonucleotide probes with sequences having SEQ ID NO: 74-82;

- mutations in the parC gene of M. genitalium S83N, S83I, S83R, D87N, D87Y, D87V, found in M. genitalium isolates resistant to fluoroquinolones. Elements with immobilized oligonucleotide probes with sequences having SEQ ID NO: 83-90;

- mutations in the parC gene of M. hominis S83I, S83N, E87K, found in M. hominis isolates resistant to fluoroquinolones. Elements are used with immobilized oligonucleotide probes with sequences having SEQ ID NO: 91-95;

- mutations in the parC gene U. urealyticum D82N, S83L, found in U. urealyticum isolates resistant to fluoroquinolones. Elements are used with immobilized oligonucleotide probes with sequences having SEQ ID NO: 96-99;

- mutations in the ntr4 _tv C213G gene (Y71STOP) and the ntr6 _tv A238T gene (K80STOP) of T. vaginalis, found in T. vaginalis isolates resistant to nitroimidazoles. Elements with immobilized oligonucleotide probes with sequences having SEQ ID NO: 100-103;

- the presence in the sample of the analyzed DNA of the nimB, nimC, nimD, nimE, nimF, nimG genes associated with the resistance of the microorganism (s) to nitroimidazoles. Elements with immobilized oligonucleotide probes with sequences having SEQ ID NO: 104-109;

- the presence in the sample of the analyzed DNA plasmids bla _SHV-12 bla _LEN-17 , which characterizes the resistance of the microorganism (s) to penicillin antibiotics and cephalosporins. An element with an immobilized oligonucleotide probe with a sequence having SEQ ID NO: 110;

- the presence in the sample of the analyzed DNA plasmid bla _SHV-97 , which characterizes the resistance of the microorganism (s) to penicillin antibiotics. An element with an immobilized oligonucleotide probe with a sequence having SEQ ID NO: 111;

- the presence in the sample of the analyzed DNA plasmids bla _TEM bla _SHV , which characterizes the resistance of the microorganism (s) to penicillin antibiotics, as well as mutations in the plasmid bla _TEM M182T and G238S, associated with resistance to penicillin antibiotics and cephalosporins. Elements with immobilized oligonucleotide probes with sequences having SEQ ID NO: 112-117;

- a mutation in the penA gene of N. gonorrhoeae: an insertion of aspartic acid D into position 345, found in N. gonorrhoeae isolates resistant to penicillin antibiotics. Elements with immobilized oligonucleotide probes with sequences having SEQ ID NO: 118-119;

- a mutation in the ponA gene of N. gonorrhea L421P, which is found in penicillin-resistant antibiotic-resistant N. gonorrhoeae isolates. Elements with immobilized oligonucleotide probes with sequences having SEQ ID NO: 120-123;

- mutations in the rpsJ gene of N. gonorrhoeae V57M, V57L, found in tetracycline-resistant N. gonorrhoeae isolates. Elements with immobilized oligonucleotide probes with sequences having SEQ ID NO: 124-126;

- the presence in the sample of the analyzed DNA of the tetM gene, which is found in isolates of E. faecalis, E. coli, M. genitalium, M. hominis, N. gonorrhoeae, G. vaginalis, resistant to tetracyclines. Elements with immobilized oligonucleotide probes with sequences having SEQ ID NO: 127-128;

- the presence in the sample of the analyzed DNA of the tetO gene found in E. faecalis, S. epidermidis isolates resistant to tetracyclines. An element with an immobilized oligonucleotide probe with a sequence having SEQ ID NO: 129;

- mutations in 23S RNA of U. urealyticum, U. hominis, S. anginosus, F. nucleatum, E. faecali, T. pallidum, E. coli, N. gonorrhoeae, C. trachomatis, M. genitalium, M. hominis, S epidermidis, B. fragilis G2057A, A2058G, A2058C, A2059G, found in isolates of the listed pathogens resistant to macrolides. Elements with immobilized oligonucleotide probes with sequences having SEQ ID NO: 130-139;

- the presence in the sample of the analyzed DNA of the pump gene efflux mefA S. anginosus, N. gonorrhoeae, S. epidermidis, B. fragilis, which leads to the resistance of S. anginosus, N. gonorrhoeae, S. epidermidis, B. fragilis to macrolides. Elements with immobilized oligonucleotide probes with sequences having SEQ ID NO: 140-141;

- mutations in 16S N. gonorrhoeae С1192Т, G1064C RNA, found in spectinomycin-resistant N. gonorrhoeae isolates. Elements with immobilized oligonucleotide probes with sequences having SEQ ID NO: 142-150;

- mutations in 16S RNAs of C. trachomatis, E. coli, F. nucleatum, A. vaginae, T. pallidum, U. urealyticum, U. hominis, S. anginosus, S. epidermidis, M. genitalium, M. hominis, M mulieris, B. fragilis G1058C, G1064C, found in isolates of listed pathogens resistant to tetracycline. Elements with immobilized oligonucleotide probes with sequences having SEQ ID NO: 148, 151-155;

- mutations in the pump efflux mtrR gene: deletion of adenine A at position -35 of the promoter region or insertion of thymidine T or TT at position -10, found in isolates of pathogens resistant to penicillin antibiotics, tetracyclines, macrolides, cephalosporins. Elements with immobilized oligonucleotide probes with sequences having SEQ ID NO: 156-161;

- mutation in the porIn protein gene of porB N. gonorrhoeae G120K, which is found in N. gonorrhoeae isolates resistant to penicillin-type antibiotics, cephalosporins, tetracyclines. Elements with immobilized oligonucleotide probes with sequences having SEQ ID NO: 162-163 are used.

When choosing oligonucleotide probes for immobilization on a DNA chip, the size and complexity of the analyzed sequence, in particular, the presence of repeats and extended homopolymer sequences, are taken into account, and the length of the discriminating oligonucleotides is determined to ensure their specificity with respect to the analyzed sequence. For each position for which mutations or a species-specific single nucleotide polymorphism is known, a set of specific discriminating oligonucleotides capable of identifying known variants of substitutions is selected. Using software such as Oligo v. 6.3 (Molecular Biology Insights Inc., USA), the melting points of oligonucleotides are calculated and, by varying their length, they ensure that the melting range of oligonucleotides is no more than 2–3 ° C. Avoid such oligonucleotides that are capable of forming secondary hairpin-type structures with high melting points. The position of the variable nucleotides and other nucleotide rearrangements to be determined is chosen, if possible, no further than 1-4 nucleotides from the middle of the corresponding discriminating oligonucleotide.

Procedure for the identification of genetic determinants antimicrobial agents IPNRF antimicrobial using a DNA chip include (a) providing a clinical specimen (scraping material from the mucosa of the urogenital system), (b) PCR amplification using oligonucleotide primers complementary to the corresponding DNA fragments pathogens , (c) hybridization of the obtained PCR products on DNA chips, (d) registration and interpretation of hybridization results.

PCR amplification is performed by any method that allows to obtain DNA fragments corresponding to the analyzed sequences, for example, as described in previously published works: Zimenkov DV, Kulagina EV, Antonova OV, Surzhikov SA, Bespyatykh Yu.A., Shitikov E.A., Ilina E.N., Mikhailovich V.M., Zasedatelev A.S. Gryadinov D.A. Analysis of the genetic determinants of multiple and broad drug resistance of the causative agent of tuberculosis using an oligonucleotide microchip. Molecular Biology, 2014, vol. 48 (2), p. 251-264; Garyadov D.A., Getman I.A., Chizhova S.N., Mikhailovich V.M., Zasedatelev AS, Romanov G.A. Identification of genetically modified sources of plant origin in food products and raw materials using a hydrogel oligonucleotide microchip. Molecular Biology, 2011, vol. 45 (6), p. 973-983; Zimenkov DV, Kulagina EV, Antonova OV, Zhuravlev VY, Gryadunov DA. 2016. Simultaneous drug resistance and Mycobacterium tuberculosis using a low-density hydrogel microarray. Journal of Antimicrobial Chemotherapy. 71 (6): 1520-1531.

Hybridization of the obtained PCR products on a DNA chip is carried out in a solution containing a buffer component to maintain pH, salt to create ionic strength and a chaotropic (hydrogen bond destabilizing agent) in a sealed hybridization chamber at a temperature dependent on the melting temperature of the discriminating oligonucleotides immobilized on a microchip as described in Mikhailovich VM, Lapa SA, Gryadunov DA, et al. Detection of rifampicin-resistant strains of polycorase chain reaction at a TV-Microchip. Journal of Clinical Microbiology, 2001, v. 131 (1), p. 94-98.

After the hybridization is carried registration hybridization patterns and their INTERPRETATION installing analyzed DNA belonging to species of micro-organisms and pathogens IPNRF identifying genetic determinants of resistance to antimicrobials, antibiotics including penicillins, cephalosporins, tetracyclines, fluoroquinolones, macrolides, nitroimidazoles, spectinomycin.

The belonging of the analyzed DNA to the species of the microorganism, the causative agent of IPNRF, is determined by analyzing the signals of the cells of the DNA chip with immobilized oligonucleotides with SEQ ID NO: 1-33 (Fig. 1, Table 2) and comparing it with the signal value in the element of the DNA chip that does not contain oligonucleotides I ₀ (cell signal with an “O” index). If for the maximum signal in the cells of A.vaginae (SEQ ID NO: 1, 2) the ratio I _max / I ₀ ≥2 is fulfilled, then it is concluded that Atopobium vaginae DNA is present in the sample; if this ratio is fulfilled for cells of B. fragilis (SEQ ID NO: 3, 4), then it is concluded that the DNA sample Bacteroides fragilis is present in the sample, for E. coli cells (SEQ ID NO: 5, 6) - DNA of Escherichia coli and t .d If for a pathogen the ratio is I _max / I ₀ <2, then it is concluded that there is no DNA of this pathogen in the analyzed sample, and further analysis of its antibiotic resistance is not performed.

The analysis of elements containing probes specific for mutations leading to amino acid / nucleotide substitutions is carried out by comparing the intensity of the signals of elements containing oligonucleotides corresponding to the wild type and mutant variants. If the maximum signal is recorded in the element corresponding to the wild-type pathogen DNA, it means that the analyzed DNA sample does not have mutations in this amino acid / nucleotide position. If the maximum signal in the cell corresponding to the DNA with the mutation is recorded, this means that for this amino acid / nucleotide position, the analyzed DNA sample carries a mutation associated with the resistance of the pathogen to the antimicrobial drug (s).

The presence of genetic determinants of resistance nimB, nimC, nimD, nimE, nimF, nimG, tetM, tetO, bla _TEM , bla _LEN , bla _SHV , mefA is determined by comparing the signal of the corresponding cell of the DNA chip with the signal in the element of the DNA chip that does not contain oligonucleotides I ₀ . For example, if the ratio I _nimB / I ₀ ≥2 is satisfied, it is considered that the analyzed DNA sample contains the nimB gene associated with resistance to nitroimidazoles; if the ratio I _mefA / I ₀ ≥2 is fulfilled, it is considered that the analyzed DNA sample contains the mefA gene associated with the resistance of S. anginosus, N. gonorrhoeae, S. epidermidis, B. fragilis to macrolides, etc.

The following is an algorithm for interpreting the results obtained on a DNA chip: identification of the genetic determinants of resistance of anti-microbial agents of AIPNRF.

Elements of a DNA chip for analyzing resistance to fluoroquinolones (gyrA) (Fig. 1, Table 2):

- the maximum signal from cells S91, D95 (SEQ ID NO: 34, 36) indicates the absence of mutations in the fragment of the N. gonorrhoeae gyrA gene; the maximum signal from cells F91, T91, N95, G95 (SEQ ID NO: 35, 37-42) indicates the presence in this gene of S91F, S91T, D95N, D95G mutations associated with resistance of N. gonorrhoeae to fluoroquinolones;

- the maximum signal from cells S83, D87 (SEQ ID NO: 43, 44) indicates the absence of mutations in the E. coli gyrA gene fragment; the maximum signal from cells L83, W83, A83, N87, G87, Y87 (SEQ ID NO: 47-55) indicates the presence in this gene of S83L, S83W, S83A, D87N, D87G, D87Y mutations associated with E. coli resistance to fluoroquinolones;

- the maximum signal from cell S83 (SEQ ID NO: 56) indicates the absence of mutations in the fragment of the gyrA gene of S. epidermidis; the maximum signal from cells S83, T83 (SEQ ID NO: 57, 58) indicates the presence in this gene of S83T, S83F mutations associated with S. epidermidis resistance to fluoroquinolones;

- the maximum signal from cells S83, G87 (SEQ ID NO: 59, 62) indicates the absence of mutations in the fragment of the gyrA gene of F. nucleatum; the maximum signal from cells L83, T83, R87 (SEQ ID NO: 60, 61, 63) indicates the presence in this gene of S83L, S83T, G87R mutations associated with F. nucleatum resistance to fluoroquinolones;

- the maximum signal from cell S83 (SEQ ID NO: 64) indicates the absence of mutations in the E. faecalis gyrA gene fragment; the maximum signal from T83, I83, R83 cells (SEQ ID NO: 65-67) indicates the presence of S83T, S83I, S83R mutations in this gene associated with resistance of E. faecalis to fluoroquinolones;

- the maximum signal from S83 cells (SEQ ID NO: 68, 69) indicates the absence of mutations in the fragment of the S. anginosus gyrA gene; the maximum signal from T83, F83 (SEQ ID NO: 70-73) cells indicates the presence of S83T, S83F mutations in this gene associated with S. anginosus resistance to fluoroquinolones.

Elements of a DNA chip for analyzing resistance to fluoroquinolones (parC (Fig. 1, Table 2):

- the maximum signal from cells S87, E91 (SEQ ID NO: 74, 78) indicates the absence of mutations in the fragment of the gene parC N. gonorrhoeae; the maximum signal from cells N87, R87, Q91, G91, K91, A91 (SEQ ID NO: 75-77, 79-82) indicates the presence of mutations S87N, S87R, E91Q, E91G, E91K, E91A associated with resistance in this gene N. gonorrhoeae to fluoroquinolones;

- the maximum signal from the cells S83, D87 (SEQ ID NO: 83, 87) indicates the absence of mutations in the fragment of the gene parC M. genitalium; the maximum signal from cells N83, I83, R83, N87, Y87, V87 (SEQ ID NO: 84-86, 88-90) indicates the presence of mutations in this gene of S83N, S83I, S83R, D87N, D87Y, D87V associated with resistance M. genitalium to fluoroquinolones;

- the maximum signal from the cells S83, E87 (SEQ ID NO: 91, 93) indicates the absence of mutations in the fragment of the parC gene M. hominis; the maximum signal from cells I83, N83, K87 (SEQ ID NO: 92, 94, 95) indicates the presence in this gene of S83I, 83N, E87K mutations associated with M. hominis resistance to fluoroquinolones;

- the maximum signal from cell D82 S83 (SEQ ID NO: 96) indicates the absence of mutations in the fragment of the parC U. urealyticum gene; the maximum signal from cells N82, L83 (SEQ ID NO: 97-99) indicates the presence in this gene of D82N, S83L mutations associated with U. urealyticum resistance to fluoroquinolones;

Elements of a DNA chip for analyzing resistance to nitroimidazoles (ntr4 _tv , ntr6 _tv ) (Fig. 1, Table 2):

- the maximum signal from cells 213C, 238A (SEQ ID NO: 100, 102) indicates the absence of mutations in the ntr4 _tv and ntr6 _tv genes, T. vaginalis; the maximum signal from cells 213G, 238T (SEQ ID NO: 101, 103) indicates the presence of C213G mutations (Y71STOP) in this gene in the ntr4 _tv gene and A238T (K80STOP) in the ntr6 _tv gene found in T. vaginalis isolates resistant to nitroimidazole.

Elements of a DNA chip for analyzing resistance to nitroimidazoles (nim (B-G)) (Fig. 1, Table 2):

- analyze the signals from the cells nimB, nimC, nimD, nimE, nimF, nimG (SEQ ID NO: 104-109), and when performing the ratio I _nim / I ₀ ≥2 consider that the analyzed sample of DNA contains the corresponding gene nim associated with Nitroimidazole resistance.

Elements of a DNA chip for analysis of resistance to β-lactams (bla) (Fig. 1, Table 2):

- analyze signals from the bla _SHV-12 bla _LEN-17 cell (SEQ ID NO: 110), and when performing the ratio I _bla / I ₀ ≥2, it is considered that the analyzed DNA sample contains the bla gene _SHV-12 and / or bla _{LEN- 17} associated with resistance to penicillin antibiotics and cephalosporins;

- analyze the signals from the bla _SHV-97 cell (SEQ ID NO: 111), and when performing the ratio I _bla / I ₀ ≥2, it is considered that the analyzed DNA sample contains the bla gene _SHV-97 associated with resistance to the antibiotics of the penicillin series;

- analyze signals from bla cells _TEM , bla _SHV , M182, T182, G238, S283 (SEQ ID NO: 112-117), and when performing the ratio I _bla / I ₀ ≥2 consider that the analyzed sample of DNA contains the corresponding gene associated with antibiotic resistance to penicillin; at the same time, the maximum signal from T182 and / or S283 cells indicates the presence of substitutions M182T and / or G238S, indicating resistance to penicillin-type antibiotics and cephalosporins

Elements of a DNA chip for analysis of resistance to β-lactams (penA, ponA) (Fig. 1, Table 2):

- the maximum signal from the penA wt cell (SEQ ID NO: 118) indicates the absence of mutations in the fragment of the penA gene of N. gonorrhoeae; the maximum signal from the insD cell (SEQ ID NO: 119) indicates the presence of aspartic acid (D) insertion in this gene at position 345 found in N. gonorrhoeae isolates resistant to penicillin antibiotics;

- the maximum signal from cells L421 (SEQ ID NO: 120, 121) indicates the absence of mutations in the fragment of the ponA gene of N. gonorrhoeae; the maximum signal from cell P421 (SEQ ID NO: 122, 123) indicates the presence in this gene of the L421P mutation associated with the resistance of N. gonorrhoeae to penicillin antibiotics.

Elements of a DNA chip for tetracycline resistance analysis (rpsJ) (Fig. 1, Table 2):

- the maximum signal from cell V57 (SEQ ID NO: 124) indicates the absence of mutations in the rpsJ gene fragment of N. gonorrhoeae; the maximum signal from cell M57, L57 (SEQ ID NO: 125, 126) indicates the presence in this gene of V57M, V57L mutations associated with N. gonorrhoeae resistance to tetracyclines.

Elements of a DNA chip for tetracycline resistance analysis (tetM, tetO) (Fig. 1, Table 2):

- analyze signals from tetM cells, tetO cells (SEQ ID NO: 127-129), and when performing the ratio I _tet / I ₀ ≥2, it is considered that the analyzed DNA sample contains the corresponding gene associated with resistance to tetracyclines (tetM in E. faecalis , E. coli, M. genitalium, M. hominis, N. gonorrhoeae, tetO in E. faecalis isolates, S. epidermidis).

Elements of a DNA chip for analysis of resistance to macrolides (23S RNA) (Fig. 1, Table 2):

- the maximum signal from cell 2058A (SEQ ID NO: 130) indicates the absence of mutations in pos. 257-259 23S U. urealyticum RNA, U. hominis, S. anginosus, F. nucleatum, E. faecalis; the maximum signal from cells 2058G, 2058C, 2059G (SEQ ID NO: 132-134, 138) indicates the presence of substitutions A2058G, A2058C, A2059G, G2057A, found in isolates of the listed pathogens resistant to macrolides;

- the maximum signal from cell A2058 (SEQ ID NO: 131) indicates the absence of mutations in pos. 257-259 23S RNAs of T. pallidum, E. coli, N. gonorrhoeae, C. trachomatis, M. genitalium, M. hominis, S. epidermidis, B. fragilis; the maximum signal from cells 2058G, 2058C, 2059G, 2057A (SEQ ID NO: 135-137, 139) indicates the presence of substitutions A2058G, A2058C, A2059G, G2057A, occurring in isolates of the listed pathogens resistant to macrolides.

Elements of a DNA chip for analysis of resistance to macrolides (mef) (Fig. 1, Table 2):

- analyze signals from cells mefA (SEQ ID NO: 140, 141), and when performing the ratio I _mef / I ₀ ≥2 believe that the analyzed DNA sample contains the mefA gene associated with resistance S. anginosus, N. gonorrhoeae, S. epidermidis, V. fragilis to macrolides.

Elements of a DNA chip for analysis of resistance to tetracyclines, spectinomycin (16S RNA) (Fig. 1. Table 2):

- the maximum signal from cells 1192C, 1064G-1058G (SEQ ID NO: 142-144, 148) indicates the absence of mutations in the 16S RNA of N. gonorrhoeae; the maximum signal from cells 1064C-1058G, 1064C-1058C (SEQ ID NO: 145-147, 149-150) indicates the presence of nucleotide substitutions C1192T, G1064C, associated with resistance of N. gonorrhoeae to spectinomycin.

- the maximum signal from cells 1064G-1058G (SEQ ID NO: 148, 151) indicates the absence of mutations in the 16S RNA of C. trachomatis, E. coli, F. nucleatum, A. vaginae, T. pallidum, U. urealyticum, U. hominis, S. anginosus, S. epidermidis, M. genitalium, M. hominis, M. mulieris, B. fragilis; the maximum signal from cells 1064G-1058C, 1064C-1058G, 1064C-1058C (SEQ ID NO: 152-155) indicates the presence of nucleotide substitutions G1058C, G1064C found in isolates of the listed pathogens resistant to tetracycline.

The elements of the DNA chip for analyzing the gene efflux mtrR gene (Fig. 1, Table 2):

- the maximum signal from cells -35A, -10A (SEQ ID NO: 156, 158, 161) indicates the absence of mutations in the efflux pump mtrR gene; maximum cell signal -35 delA,

insT, insTT (SEQ ID NO: 157, 159-160) indicates the presence of mutations in the mtrR gene associated with resistance to penicillin antibiotics, tetracyclines, macrolides, cephalosporins.

Elements of a DNA chip for analyzing the porB porin protein gene (Fig. 1, Table 2):

- the maximum signal from cell G120 (SEQ ID NO: 162) indicates the absence of mutations in the porB gene of N. gonorrhoeae; the maximum cell signal of D120 indicates the presence of a mutation G120K, which is found in N. gonorrhoeae isolates resistant to penicillin antibiotics, cephalosporins, tetracyclines.

The described analysis algorithm can be implemented in software that allows for automatic registration and interpretation of analysis results using a DNA chip.

Examples

Example 1. Manufacture of a DNA chip to identify genetic determinants of antibiotic resistance of infectious agents that lead to impaired human reproductive functions

The synthesis of oligonucleotides with the sequences SEQ ID NO: 1-163 (Table 1) for immobilization on a DNA chip was performed on an automatic ABI-394 DNA / RNA synthesizer synthesizer (Applied Biosystems, USA) using the standard phosphoramidite method and purified using the reversed method phase HPLC (Gilson complex, France). In the course of the synthesis, a free amino group spacer was introduced into oligonucleotides using 5'-Amino-Modifier C6 (Glen Research, USA). DNA chips were manufactured according to the procedure described previously (Rubina AY, Pan'kov SV et al. Hydrogel drop microchips with immobilized production. Anal. Biochem., 2004, v. 325, p. 92-106).

Polymerization mixtures containing a mixture of gel-forming monomers (derivatives of methacrylamide, methylene bis-acrylamide, stabilizers, etc.) and immobilizable compounds (oligonucleotides or fluorescent IMD 504 dye) were placed in cells of a 384-well plate. Using a QArray robot (Genetix, UK), the mixtures from the plate wells were transferred in the form of microdroplets onto the surface of plastic substrates. Polymerization of the gel with simultaneous covalent immobilization of oligonucleotides in the gel structure occurred under the action of UV radiation (UV lamp Sylvania GTE F15T8350 BL, United Kingdom), 40 min, 20 ° C, in a stream of nitrogen. After polymerization, the substrates with arrays of hydrogel cells (microdroplets) were washed with 0.1 M phosphate buffer, water and dried in a dustless atmosphere.

A diagram of a hydrogel-based DNA chip for identifying the genetic determinants of antibiotic resistance of the causative agents of IPNRF is shown in FIG. 1. The location of the oligonucleotide probes immobilized on the DNA chip in accordance with the SEQ ID is given in Table 2.

Example 2. Identification of genetic determinants of resistance of anti-microbial agents to IPNRF using a DNA chip

The analysis procedure included the following stages: isolation of DNA from clinical material, multiplex PCR using a set of specific oligonucleotide primers and a mixture of four deoxynucleoside triphosphates, one of which contained a fluorescent label, to obtain fluorescently labeled DNA fragments; hybridization of amplified fluorescently-labeled products on DNA chips; registration of fluorescent images (hybridization pictures) and interpretation of the results of hybridization.

a) Processing of clinical material and DNA extraction

Processing samples of clinical material - scrapings from the mucous membrane of the organs of the urogenital system, and the isolation of total DNA was performed using a set of DNA Express for DNA extraction from biological samples (ZAO Litekh, Russia) according to the manufacturer's recommendations.

b) Preparation of primers for multiplex PCR

When selecting primer sequences for PCR, a database of nucleotide sequences was used, known to specialists in the field, for example, the database http://www.ncbi.nlm.nih.gov/Genbank/index.html. The specificity of the primers was checked using software that uses a search in the bases of nucleotide sequences using the BLAST algorithm (www.ncbi.nlm.nih.gov/BLAST). The list of primers used in this example is shown in Table 3. The primers were synthesized on an automatic ABI-394 DNA / RNA synthesizer synthesizer (Applied Biosystems, USA) using the standard phosphoramidite method and purified using reverse phase HPLC (Gilson complex , France).

Legend: f - direct primer, r - reverse primer,

R = G or A; Y = T or C; K = G or T.

c) Conducting multiplex PCR

Prepared two mixtures of primers for PCR PR-1 and PR-2, containing oligonucleotides listed in Table 3. The number of each primer in the reaction mixture is: for a mixture of PR-1 - 10 nmol (forward primer), 50 nmol (reverse primer) , for a mixture of PR-2 - 40 nmol (direct primer), 200 nmol (reverse primer). Two reaction mixtures were prepared for PCR, introducing the following reagents into the tube (based on 1 reaction).

Mix 1:

- 10 × PCR buffer for HotStarTaq DNA polymerase, Qiagen, Germany 3 μl; - dNTP (aqueous solution of deoxynucleoside triphosphates, 2 mM each) 3 μl; - fluorescently labeled substrate, 1 mm 0.2 μl - a mixture of primers PR-1 1.0 µl; - HotStarTaq DNA polymerase, Qiagen, Germany 1.0 µl; - water is deionized 18 μl

Mix 2:

- 10 × PCR buffer for HotStarTaq DNA polymerase, Qiagen, Germany 3 μl; - dNTP (aqueous solution of deoxynucleoside triphosphates, 2 mM each) 3 μl; - fluorescently labeled substrate, 1 mm 0.2 μl - a mixture of primers PR-2 1.0 µl; - HotStarTaq DNA polymerase, Qiagen, Germany 1.0 µl; - water is deionized 18 μl

As a fluorescently labeled substrate, a dUTP conjugate with a fluorescent dye was used (absorption maximum at 645 nm, fluorescence maximum at 670 nm) (Kuznetsova VE, Spitsyn MA, Shershov VE, Guseinov T.O., Fesenko EE, Lapa SA, Ikonnikova A.Iu., Avdonina MA, Nasedkina TV, Zasedatelev AS, Chudinov AV Novel fluorescently labeled nucleotides chain reaction, Mendeleev Communications, 2016, v. 26 (2), p. 95-98 ). The fluorescent substrate is inserted by DNA polymerase into the growing DNA strand during PCR.

To the obtained PCR mixtures were added 1 μl of the DNA solution obtained from the clinical sample. For the negative control, 1 μl of deionized water was used. Multiplex PCR was performed on a S1000 amplifier (Bio-Rad, USA, using the temperature-time mode given below (Table 4).

Solutions after carrying out PCR were used to conduct hybridization on a DNA chip, manufactured as described in Example 1.

d) Hybridization of fluorescently-labeled products on DNA chips

10 μl of hybridization buffer (0.3 M HEPES, pH 7.5, 3.0 M guanidine thiocyanate, 30 mM EDTA) and 10 μl of reaction mixtures after PCR were added to the tube. 30 μl of the resulting hybridization mixture was placed in the reaction chamber of a DNA chip and hybridization was performed at 37 ° C for 6-12 hours. At the end of the hybridization, the DNA chip was washed three times with distilled water at 37 ° C, the hybridization chamber was removed, and the DNA chip was dried in an air stream,

e) Registration and interpretation of hybridization results on DNA chips

The registration of fluorescent images of DNA chips (hybridization pictures) was performed on a universal hardware-software complex (UAPK) (BIOCHIP-IMB LLC, Russia) for analyzing biological microchips using the specialized software Image Ware® (BIOCHIP-IMB software) . Hybridization patterns are shown in FIG. 2-22.

In accordance with the algorithm for interpreting the results obtained on the DNA chip described above, it was established that the analyzed DNA belongs to the species of microorganisms that are the causative agents of the AIPNRF (T. pallidum, N. gonorrhoeae, C. trachomatis, M. genitalium, M. hominis, G. vaginalis , U. parvum, U. urealyticum, A. vaginae, E. faecalis, T. vaginalis, E. coli, M. mulieris, S. anginosus, F. nucleatum, S. epidermidis, B. fragilis), and determined the presence of genetic determinant of resistance to antimicrobial drugs, including penicillin antibiotics, cephalosporins, tetracyclines, fluoroquinolones, macrolides, nitroimidazoles, spectra omitsin

The correct identification of genetic determinants of resistance of pathogens IPRNF to antimicrobial drugs using the proposed DNA chip in all cases checked by sequencing the corresponding fragments of the genome of the pathogen. Sequencing was performed on a 3130xL Applied Biosystems (USA) automatic analyzer using the BigDye Terminator v3.1 Ready Reaction Cycle Sequencing Kit. The results obtained using the DNA chip proposed in this invention and the reference method coincided for all analyzed samples.

Thus, the DNA chip presented in this invention is a tool for carrying out multiplex analysis, which allows for a short time to accurately determine the genetic determinants of resistance of the causative agents of AIPNRF to current antibacterial drugs in clinical material. The DNA chip compares favorably with the currently available methods for determining antibiotic resistance by the possibility of simultaneous analysis of 17 pathogens of AIPNRF, a wide range of simultaneously detectable determinants of resistance, and the speed of analysis. DNA chip applications include medicine, including dermatovenereology, urology, gynecology, clinical laboratory diagnostics, and epidemiology.

Claims (34)

1. DNA chip for identifying genetic determinants of microbial resistance - pathogens leading to the violation of human reproductive functions: Treponema pallidum, Neisseria gonorrhoea, Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Gardnerella vaginalis, urethra, urengro faecalus, trichomonos, The binder in that it is a matrix of cells 168 placed on a substrate made of glass or plastic, wherein the matrix comprises: - 33 cells with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 1-33, corresponding to 16S rRNAs of T. pallidum, N. gonorrhoeae, C. trachomatis, M. genitalium, M. hominis, G. vaginalis, U. parvum, U. urealiticum, A. vaginae, E. faecalis, T. vaginalis, E. coli, M. mulieris, S. anginosus, F. nucleatum, S. epidermidis, B. fragilis, to establish the identity of the analyzed DNA to the above types of microorganisms that are pathogens that lead to impaired human reproductive functions; - 9 cells with immobilized oligonucleotides with nucleotide sequences of SEQ ID nos: 34-42, corresponding to the sequences of the gyrA gene of wild-type N. gonorrhoeae isolates or sequences of mutant variants of the gyrA gene, resulting in S91F, S91T, D95N, D95G amino acid substitutions, which were found in chyra, and the sequences of the gene samples have been obtained. Fluoroquinolone-resistant gonorrhoeae; - 13 cells with immobilized oligonucleotides with nucleotide sequences of SEQ ID NO: 43-55, corresponding sequences of the wild-type E. coli gyrA gene, or sequences of mutant gyrA gene variants leading to amino acid substitutions S83L, S83W, S83A, D87N, D87G, D87Y, found in fluoroquinolone-resistant isolates; - 3 cells with immobilized oligonucleotides with the nucleotide sequences SEQ ID NO: 56-58, corresponding to sequences gyrA gene S. epidermidis isolates of wild-type or mutant sequences embodiment gyrA gene leading to amino acid substitutions S83T, S83F, occurring in S. epidermidis isolates, resistant to fluoroquinolones; - 5 cells with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 59-63, corresponding to the gyrA gene sequences of wild-type F. nucleatum isolates or the sequences of mutant gyrA gene, leading to amino acid substitutions S83L, S83T, G87R, found in the phytotheme isolates resistant to fluoroquinolones; - 4 cells with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 64-67, corresponding to the sequences of the gyrA gene of wild-type E. faecalis isolates or sequences of mutant variants of the gyrA gene, leading to amino acid substitutions S83T, S83I, S83R found in E. resistant to fluoroquinolones; - 6 cells with immobilized oligonucleotides with the nucleotide sequences of SEQ ID 68-73, the respective gene sequences gyrA isolates S. anginosus wild-type or mutant sequences embodiment gyrA gene leading to amino acid substitutions S83T, S83F, occurring in isolates S. anginosus, resistant to fluoroquinolones ; - 9 cells with immobilized oligonucleotides with nucleotide sequences of SEQ ID NO: 74-82, corresponding sequences of the wild-type parC gene of N. gonorrhoeae isolates or sequences of mutant variants of the parC gene, leading to amino acid substitutions for S87N, S87R, E91Q, E91G, E91K, E91A, found in the fluoroquinolone-resistant isolates; - 8 cells with immobilized oligonucleotides with the nucleotide sequences SEQ ID NO: 83-90, corresponding gene sequences parS M. genitalium isolates of wild-type or mutant sequences embodiment parC gene leading to amino acid substitutions S83N, S83I, S83R, D87N, D87Y, D87V, found in M. genitalium isolates resistant to fluoroquinolones; - 5 cells with immobilized oligonucleotides with nucleotide sequences of SEQ ID NOS: 91-95, corresponding to the sequences of the parC gene of wild-type M. hominis isolates or the sequences of mutant parC gene, leading to amino acid substitutions S83I, S83N, E87K, found in the hominis M isolates, fluoroquinolone resistant; - 4 cells with immobilized oligonucleotides with nucleotide sequences of SEQ ID NO: 96-99, corresponding to the sequence of the gene patterns of the wild-type U. urealiticum isolate gene or the sequences of the mutant parC gene, leading to amino acid substitutions D82N, S83L, found in the isolates of U. urealiticumumumumus uumiticumus, which are found in U. urealiticum amino acid substitutions found in U. urealiticum gene isolates. to fluoroquinolones; - 4 cells with immobilized oligonucleotides with the nucleotide sequences SEQ ID NO: 100-103, the relevant gene sequences ntr4 _tv and ntr6 _tv T. vaginalis isolates of wild-type sequences or their mutated variants with nucleotide substitutions C213G (Y71STOP) gene ntr4 _tv, A238T ( K80STOP) in the ntr6 _tv gene found in T. vaginalis isolates resistant to nitroimidazoles; - 6 cells with immobilized oligonucleotides with nucleotide sequences of SEQ ID NO: 104-109, corresponding sequences of genes nimB, nimC, nimD, nimE, nimF, nimG, the presence of which leads to the resistance of the microorganism (s) to nitroimidazoles; - a cell with an immobilized oligonucleotide with the nucleotide sequence of SEQ ID NO: 110, corresponding to the sequence of the _SHV-12 bla _LEN-17 plasmid genes, the presence of which leads to resistance of the microorganism (s) to penicillin antibiotics and cephalosporins; - a cell with an immobilized oligonucleotide with the nucleotide sequence of SEQ ID NO: 111, corresponding to the sequence of the plasmid gene _SHV-97 bla, the presence of which leads to the resistance of the microorganism (s) to penicillin antibiotics; - 6 cells with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 112-117, corresponding to the sequences of the bla _TEM and bla _SHV plasmid genes, the presence of which leads to resistance of the microorganism (s) to antibiotics of the penicillin series, or the sequences of the mutant variant of the bla _TEM gene with amino acids substitutions M182T and G238S, the simultaneous presence of which leads to the resistance of the microorganism (s) to penicillin antibiotics and cephalosporins; - 2 cells with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 118-119, corresponding to the sequences of the penA gene of wild-type N. gonorrhoeae isolates or sequences of the mutant variant of the penA gene, resulting in an insertion of aspartic acid (D) at position 345 found in isolates N Penicillin-resistant antibiotic-resistant gonorrhoeae; - 4 cells with immobilized oligonucleotides with nucleotide sequences of SEQ ID NO: 120-123, corresponding to the sequences of the ponA gene of wild-type N. gonorrhoeae isolates, or the sequences of the mutant variant of the ponA gene with the amino acid substitution L421P found in penicillin-resistant antibiotic-resistant N. gonorrhoeae isolates; - 3 cells with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 124-126, corresponding to the sequences of the rpsJ gene sequences of wild-type N. gonorrhoeae gene isolates or sequences of the mutant version of the rpsJ gene with the amino acid substitution V57M, V57L, found in the gates of N. gonorrhoe gene, which are in amino acid substitutions of V57M, V57L, found in the gates of N. gonorrhoe gene, which are in amino acid substitutions of the N. gonorrhoeae gene, found in the N. ; - 2 cells with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 127-128, corresponding to the sequences of the tetM genes, whose presence in E. faecalis, E. coli, M. genitalium, M. hominis, N. gonorrhoeae, G. vaginalis leads to tetracycline resistance; - a cell with an immobilized oligonucleotide with the nucleotide sequence of SEQ ID NO: 129, corresponding to the tetO gene sequence, whose presence in E. faecalis, S. epidermidis isolates leads to tetracycline resistance; - 10 cells with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 130-139, corresponding to the sequences of 23S RNA isolates from U. urealiticum, U. hominis, S. anginosus, F. nucleatum, E. faecali, T. pallidum, E. coli, N. gonorrhoeae, C. trachomatis, M. genitalium, M. hominis, S. epidermidis, Wild B. fragilis or sequences of mutant variants with G2057A, A2058G, A2058C, A2059G substitutions found in isolates of the listed pathogens resistant to macrolides; - 2 cells with immobilized oligonucleotides with nucleotide sequences SEQ ID NO: 140-141, corresponding to the mefA gene sequence, the presence of which leads to the resistance of S. anginosus, N. gonorrhoeae, S. epidermidis, B. fragilis to macrolides; - 9 cells with immobilized oligonucleotides with nucleotide sequences of SEQ ID NO: 142-150, corresponding to the 16S RNA sequences of wild-type N. gonorrhoeae isolates or sequences of mutant variants with С1192Т, G1064C substitutes found in N. gonorrhoeae, and resistant templates, resistant templates, resistant kits, and resistant templates, churches, and resistant templates, which are resistive, they are resistive, they are used in resistant patterns, and they are resistant patterns that are used in the Ngonorrhoeae, resistant templates, G1164T, G1064C, which are found in N. gonorrhoeae isolates, resistive patterns, and resistant templates, which are used in resistant patterns, resis- tent patterns, resistant patterns, Nr gonorrhoeae, resistant patterns, resistant patterns, Nr gonorrhoeae, resistive patterns, Ngonorrhoeae, resistant numbered patterns, G1064C, are available in the Ngonorrhoeae isolates, resistive sequences of the N11 gonutrhose, N11, T, G1064C. - 6 cells with immobilized oligonucleotides with nucleotide sequences of SEQ ID NO: 148, 151-155, corresponding to the sequences of 16S RNA isolates of C. trachomatis, E. coli, F. nucleatum, A. vaginae, T. pallidum, U. urealiticum, U. hominis, S. anginosus, S. epidermidis, M. genitalium, M. hominis, M. mulieris, B. fragilis of the wild type or sequences of mutant variants with G1058C, G1064C substitutions occurring in the isolates of the listed pathogens resistant to tetracycline; - 6 cells with immobilized oligonucleotides with the nucleotide sequences SEQ ID NO: 156-161, the relevant gene sequences efflux pump mtrR wild-type or mutant sequences of the promoter region for this gene mtrR, resulting in the deletion of an adenine at position -35 A or T or insertions thymidine TT position -10, found in isolates of pathogens resistant to penicillin antibiotics, tetracyclines, macrolides, cephalosporins; - 2 cells with immobilized oligonucleotides with nucleotide sequences of SEQ ID NO: 162-163, corresponding to the gene sequence of the porin protein porB of wild-type N. gonorrhoeae isolates or the sequence of the mutant variant of the porB gene, resulting in the G120K amino acid substitution found in N. gonorrhoe isolates, in those that are in the N. to penicillin antibiotics, cephalosporins, tetracyclines. 2. A set consisting of sequences of oligonucleotide probes with SEQ ID NO 1-163 used in the DNA chip according to claim 1 for identification genetic restraints of the , Mobiluncus mulieris, Streptococcus anginosus, Fusobacterium nucleatum, Staphylococcus epidermidis, Bacteroides fragilis, antimicrobial, comprising the antibiotics penicillin, cephalosporins, tetracyclines, fluoroquinolones, macrolides, nitroimidazoles, spectinomycin.

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