RU2562866C1 - Detection method of dna of tuberculosis causative agent with simultaneous identification of its genotype and determination of genetic determinants of multiple and wide drug resistance, oligonucleotide microchip, set of primers and set of oligonucleotide probes, which are used in method - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention relates to a detection method of DNA of a tuberculosis causative agent -mycobacteria of tuberculosis complex with simultaneous identification of a genotype of the causative agent and determination of genetic determinants of resistance to a wide spectrum of antituberculous remedies, including rifampicin, isoniazid, fluoroquinolones, kanamycin, capreomicin, amikacin, ethambutol, in a clinical specimen on a differentiating oligonucleotide microchip. The method is based on a multiplex PCR with simultaneous marking of PCR products with further hybridisation of the obtained products on the oligonucleotide microchip containing a set of specific discriminating oligonucleotides. The invention also deals with an oligonucleotide microchip, a set of primers and a set of oligonucleotide probes, which are used for the method's implementation.

EFFECT: invention allows performing an analysis of DNA separated directly from a clinic specimen, detecting genotypes of a tuberculosis causative agent, which are associated with increased virulence and transmissibility, providing personified selection of effective antituberculous remedies for each certain patient and performing epidemiological genetic typing.

9 cl, 7 dwg, 3 tbl, 8 ex

Description

FIELD OF THE INVENTION

The invention relates to molecular biology, microbiology, and medicine, and provides a method for detecting tuberculosis pathogen DNA - mycobacterium tuberculosis complex (Mycobacterium tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. microti, Mycobacterium canettii, Mycobacterium caprae, Mycobacterium caprae, Mycobacterium caprae, Mycobacterium Mycobacterium mungi) with the simultaneous establishment of the pathogen genotype and determination of the genetic determinants of resistance to a wide range of anti-TB drugs, including rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin, capreomycin, ethambutol, in a clinical sample on a differentiating oligonucleotide microchip.

State of the art

The spread of M. tuberculosis resistant tuberculosis drugs is a serious global problem. Today, according to the WHO classification, pathogens with multidrug-resistant (MDR) TB, broadly drug-resistant (XDR) TB and pathogens with total (full) resistance to anti-TB drugs (super-resistant TB) are isolated. MDR-TB is caused by mycobacteria that are resistant to at least the two most effective first-line drugs, isoniazid and rifampicin. XDR-TB is a form of tuberculosis resistant to isoniazid and rifampicin, as well as to any fluoroquinolone, and to at least one of the three injectable second-line anti-TB drugs (amikacin, kanamycin or capreomycin). Drug-resistant forms of TB do not respond to standard six-month therapy with first-line anti-TB drugs and are treated for two or more years with second-line drugs that are more toxic and expensive. At the same time, treatment for drug-sensitive tuberculosis costs about $ 200, MDR-TB costs $ 5,000, and treatment for XDR-TB is even more expensive and, most importantly, takes a longer time.

In Russia, according to official statistics, in 2009 MDR-TB among newly diagnosed patients was registered in 15.4%, while the overall prevalence of MDR-strains among all TB patients in 2009 was 33.5%. Data on XDR-TB and super-resistant TB in Russia have not been published since they are not included in reporting forms (“Tuberculosis in the Russian Federation 2009” Analytical review of statistical indicators for tuberculosis used in the Russian Federation). According to the WHO, in our country, XDR strains make up about 30% of all MDR strains of M. tuberculosis. In the world, the number of cases of XDR-TB is estimated to be 25,000 per year, and almost all of them are fatal (World Health Organization, 2010. Global tuberculosis control: a short update to the 2009 report).

The spread of drug-resistant TB is facilitated by the duration of bacteriological testing of TB pathogens isolated from patients. Even using the BACTEC automatic analyzer, the procedure for assessing the sensitivity profile of a particular isolate to first-line drugs takes more than a month, while in most bacteriological laboratories, testing for sensitivity to second-line drugs (fluoroquinolones, amikacin, kanamycin, capreomycin, ethambutol, etc.) is generally not held.

In this regard, methods are needed to quickly and reliably determine the resistance of clinical strains of tuberculosis, that is, classifying tuberculosis strains as resistance classes: drug-sensitive, MDR or XDR. The results of the analysis will be directly used in clinical practice for timely correction of anti-tuberculosis therapy. The most promising methods are molecular technologies for the analysis of the genome of the causative agent of tuberculosis, which allow the identification of the genetic determinants of multidrug and broad drug resistance.

Another important clinically significant task is the genotyping of the causative agent of tuberculosis. The growing number of sequenced genomes in recent years M. tuberculosis and their subsequent in silico comparison revealed a sufficient level of genetic diversity for phylogenetically reliable constructions, at least at the level of genetic families. Analysis of the one-nucleotide polymorphism of the genomes of mycobacterium tuberculosis complex revealed five main lines - Beijing (“Beijing” line), CAS (Central Asian - “Central Asian” line), EuroAmerican (“Euro-American” line), EAI (East African-Indian - “East African-Indian” line), M. bovis (Baker L, Brown T, Maiden MC, Drobniewski F. 2004. Silent nucleotide polymorphisms and a phylogeny for Mycobacterium tuberculosis. Emerging Infectious Diseases 10: 1568-1577). To date, the prevalence of Beijing and the Euro-American lines has been observed in the territory of the Russian Federation and the countries of the former USSR, the latter, in turn, is divided into three main genotypes Ural, LAM and Haarlem (Casali N, Nikolayevskyy V, Balabanova Y, Ignatyeva O, Kontsevaya I, Harris SR, Bentley SD, Parkhill J, Nejentsev S, Hoffner SE, Horstmann RD, Brown T, Drobniewski F. Microevolution of extensively drug-resistant tuberculosis in Russia. Genome Research. 2012.22 (4): 735-45) .

It should be noted that in the Russian Federation, the Beijing genotype is the most common and occurs on average in 50% of patients (Igor Mokrousov. Insights into the origin, emergence, and current spread of a successful Russian clone of Mycobacterium tuberculosis. Clinical microbiology reviews 2013; 26 (2 ): 342-60). The establishment of the Beijing genotype is currently a clinically significant task, since its strains demonstrate important pathogenic properties, for example, increased virulence in the body of mice vaccinated with BCG (Lopez B, Aguilar D, Orozco H, Burger M, Espitia C, Ritacco V, et al. A marked difference in pathogenesis and immune response induced by different Mycobacterium tuberculosis geneotypes. Clin Exp Immunol. 2003; 133: 30-7), association with multidrug and extensive drug resistance (Mokrousov I, Narvskaya O, Vyazovaya A, Otten T, Jiao WW , Gomes LL, Suffys PN, Shen AD, Vishnevsky B. Russian "successful" clone B0 / W148 of Mycobacterium tuberculosis Beijing genotype: a multiplex PCR assay for rapid detection and global screening. Journal of Clinical Microbiology 2012 Nov 50 (11): 3757-9), ability to multiply rapidly in human macrophages and high transmissibility (Tran N Buu, Dick van Soolingen, Mai NT Huyen, Nguyen TN Lan, Hoang T Quy, Edine W Tiemersma, Kristin Kremer, Martien W Borgdorff, Frank GJ Cobelens. Increased transmission of Mycobacterium tuberculosis Beijing genotype strains associated with resistance to streptomycin: a population-based study. PLoS One 2012 13; 7 (8): e42323). The current tuberculosis epidemic in Russia is largely associated with the active spread of multi-resistant strains of the Beijing genotype and its variant B0 / W148 in the population immunized with BCG (Casali, N., Nikolayevskyy, V., Balabanova, Y., Harris, SR, Ignatyeva, O ., Kontsevaya, I., Corander, J., Bryant, J., Parkhill, J., Nejentsev, S., Horstmann, RD, Brown, T. & Drobniewski, F. Evolution and transmission of drug-resistant tuberculosis in a Russian population. Nature Genetics 2014 46, 279-86). Thus, the widespread occurrence of Beijing strains now poses a serious threat to the successful implementation of national tuberculosis control programs.

For two other families of the causative agent of tuberculosis LAM and Ural, also widely represented in the Russian Federation, no associations with high virulence and drug resistance have been found at present. This is due to the lack of molecular diagnostic tools that ensure the simultaneous determination of genetic determinants of drug resistance and genotyping of the tuberculosis pathogen.

Currently, the following molecular methods are used to identify drug-resistant strains of mycobacterium tuberculosis complex and their genotyping:

I. Real-time PCR to identify mutations responsible for drug resistance. (Real-time PCR)

Patent application WO / 2011/140237 "Process for detection of multidrug resistant tuberculosis using real-time PCR and high resolution melt analysis"

Ramirez MV, Cowart KC, Campbell PJ, Morlock GP, Sikes D, Winchell JM, Posey JE. Rapid detection of multidrug-resistant Mycobacterium tuberculosis by use of real-time PCR and high-resolution melt analysis. Journal of clinical microbiology 2010 Nov; 48 (11): 4003-9

Chen X, Kong F, Wang Q, Li C, Zhang J, Gilbert GL. Rapid detection of isoniazid, rifampin, and ofloxacin resistance in Mycobacterium tuberculosis clinical isolates using high-resolution melting analysis. Journal of clinical microbiology 2011 Oct; 49 (10): 3450-7.

Yadav R, Sethi S, Mewara A, Dhatwalia SK, Gupta D, Sharma M. Rapid detection of rifampicin, isoniazid and streptomycin resistance in Mycobacterium tuberculosis clinical isolates by high-resolution melting curve analysis. Journal of Applied Microbiology 2012 Oct; 113 (4): 856-62.

Vladimirsky M.A., Alyapkina Yu.S., Varlamov D.A., Alekseev Y.I., Shipina L.K., Shulgina M.V., Domotenko L.V., Bykadorova K.R., Gashchenko N .N., Endourova LB, Ivanova O.V., Ilyina E.A., Levkova O.A., Markova T.V., Nazemtseva V.P., Pavlova E.P., Polozov A.I. ., Shishkina N.V. Real-time PCR application to determine and control the spread of drug-resistant strains of mycobacterium tuberculosis. Tuberculosis and lung diseases. 2008.85 (4) 38-44

II. An automated procedure combining the processing of a clinical sample and DNA extraction followed by amplification with real-time detection in a separate cartridge isolated from the environment (for example, Cepheid, USA, Xpert® MTB / RIF commercial diagnostic test system and its analogues). (cartridge-based, automated diagnostic test that can identify Mycobacterium tuberculosis (MTB) and resistance to rifampicin (RIF))

Helb, D., M. Jones, E. Story, C. Boehme, E. Wallace, K. Ho, J. Kop, M.R. Owens, R. Rodgers, P. Banada, H. Safi, R. Blakemore, N.T. Lan, E.C. Jones-Lopez, M. Levi, M. Burday, I. Ayakaka, R.D. Mugerwa, B. McMillan, E. Winn-Deen, L. Christel, P. Dailey, M.D. Perkins, D.H. Persing, and D. Alland. Rapid detection of Mycobacterium tuberculosis and rifampin resistance by use of on-demand, near-patient technology. Journal of clinical microbiology 2010 48, 229-37

A list of publications is available on the WHO website at the following link:

Published evidence and commentary on the Xpert MTB / RIF assay

WHO recommendations for the use of the Xpert® MTB / RIF test system are provided in the following articles:

Rapid implementation of the Xpert MTB / RIF diagnostic test (WHO / HTM / TB / 2011.2)

Policy statement: Xpert MTB / RIF system (WHO / HTM / TB / 2011.4)

Castan, P., de Pablo, A., Fernandez-Romero, N., Rubio, JM, Cobb, BD, Mingorance, J. & Toro, C. Point-of-care system for detection of Mycobacterium tuberculosis and rifampin resistance in sputum samples. Journal of clinical microbiology 2014 52, 502-7

III. Hybridization technologies based on probes immobilized on nitrocellulose strips (“strips”) (Line probe assay)

III.I INNO-LiPA Rif.TB commercial kit by Innogenetics (Belgium)

Tortoli E, Marcelli F. Use of the INNO LiPA Rif. TB for detection of Mycobacterium tuberculosis DNA directly in clinical specimens and for simultaneous determination of rifampin susceptibility. European Journal of Clinical Microbiology and Infectious Diseases 2007 Jan; 26 (1): 51-5.

Skenders GK, Holtz TH, Riekstina V, Leimane V. Implementation of the INNO-LiPA Rif. TB® line-probe assay in rapid detection of multidrug-resistant tuberculosis in Latvia. International Journal of Tubercle and Lung Diseases 2011 Nov; 15 (11): 1546-52

III.II GenoType ^® MTBDRplus and GenoType ^® MTBDRsl commercial kits from Hain Lifescience (Germany)

The kits are based on patent application WO / 2003/039703 "Method in the form of a dry rapid test for detecting nucleic acids" by Hain Lifescience.

Hillemann D, Rüsch-Gerdes S, Richter E. Evaluation of the GenoType MTBDRplus assay for rifampin and isoniazid susceptibility testing of Mycobacterium tuberculosis strains and clinical specimens. Journal of clinical microbiology 2007 Aug; 45 (8): 2635-40.

Nikolayevskyy V, Balabanova Y, Simak T, Malomanova N, Fedorin I, Drobniewski F. Performance of the Genotype MTBDR Plus assay in the diagnosis of tuberculosis and drug resistance in Samara, Russian Federation. BMC Clinical Pathology. 2009 Mar 10; 9: 2.

Miotto P, Cabibbe AM, Mantegani P, Borroni E, Fattorini L, Tortoli E, Migliori GB, Cirillo DM. GenoType MTBDRsl performance on clinical samples with diverse genetic background. European Respiratory Journal. 2012 Sep; 40 (3): 690-8

Barnard M, Warren R, Van Pittius NG, van Helden P, Bosman M, Streicher E, Coetzee G, O'Brien R. GenoType MTBDRsl Line Probe Assay Shortens Time to Diagnosis of XDR-TB in a High-throughput Diagnostic Laboratory. American Journal of Respiratory and Critical Care Medicine. 2012 Oct 18

III.III Commercial AID TB resistance line probe assay kits from Autoimmun Diagnostika GmbH (AID) (Germany).

B. Molina-Moya, A. Lacoma, C. Prat, J. Diaz, A. Dudnyk, L. Haba, J. Maldonado, S. Samper, J. Ruiz-Manzano, V. Ausina, J. Dominguez. AID TB resistance line probe assay for rapid detection of resistant Mycobacterium tuberculosis in clinical samples. Journal of Infection 2014. In press. DOI: http://dx.doi.org/10.1016/j.jinf.2014.09.09.011

Ritter C, Lucke K, Sirgel FA, Warren RW, van Helden PD, Böttger EC, Bloemberg GV. Evaluation of the AID TB resistance line probe assay for rapid detection of genetic alterations associated with drug resistance in Mycobacterium tuberculosis strains. Journal of clinical microbiology 2014 Mar; 52 (3): 940-6

IV. Hybridization technologies based on oligonucleotide microarrays (DNA microarrays (biochips)).

IV.I Hydrogel oligonucleotide microchips (biochips) of low density. Commercial kits “TB-Biochip-1” and “TB-BIOCHIP-2”, developed at the Institute of Molecular Biology named after V.A. Engelhardt RAS, to identify strains of the causative agent of tuberculosis and determine its sensitivity to rifampicin / isoniazid and fluoroquinolones.

Mirzabekov A.D., Mikhailovich V.M., Sobolev A.Yu. Gryadunov D.A., Lapa S.A., Zasedatelev A.S. A method for the simultaneous detection of mycobacterium tuberculosis complex and the identification of mutations in mycobacterial dna, leading to the resistance of microorganisms to rifampicin and isoniazid, on biological microchips, a set of primers, a biochip and a set of oligonucleotide probes used in the method. RF patent 2376387. Published on December 20, 2009. Priority dated 12/26/2005

Gryadunov D., V. Mikhailovich, S. Lapa, N. Roudinskii, M. Donnikov, S. Pan'kov, O. Markova, A. Kuz'min, L. Chernousova, O. Skotnikova, A. Moroz, A. Zasedatelev and A. Mirzabekov. Evaluation of hybridization on oligonucleotide microarrays for analysis of drug-resistant Mycobacterium tuberculosis. Clinical microbiology and infection 2005. Vol 11: p. 531-539.

O.V. Antonova, D.A. Gryadunov, S.A. Lapa, A.V. Kuzmin, E.E. Larionova, T.G. Smirnova, E.Yu. Nosova, O.I. Skotnikova, L.N. Chernousova, A.M. Frost, A.S. Zasedatelev, V.M. Mikhailovich. Identification of mutations in the genome of Mycobacterium tuberculosis leading to resistance to fluoroquinolones by hybridization on biological microarrays. Bulletin of Experimental Biology and Medicine 2008. No. 1. p. 115-120

Zimenkov DV, Antonova OV, Kuz Min AV, Isaeva YD, Krylova LY, Popov SA, Zasedatelev AS, Mikhailovich VM, Gryadunov DA. Detection of second-line drug resistance in mycobacterium tuberculosis using oligonucleotide microarrays. BMC Infectious Diseases. 2013 May 24; 13 (1): 240.

Zimenkov D.V., Kulagina E.V., Antonova O.V., Surzhikov S.A., Bespyatykh Yu.A., Shitikov E.A., Ilyina E.N., Mikhailovich V.M., Zasedatelev A .S., Gryadunov D.A. Analysis of the genetic determinants of multiple and wide drug resistance of the causative agent of tuberculosis using an oligonucleotide microchip. 2014. Molecular biology. t. 48 (2), pp. 251-264

IV.II Other low-density oligonucleotide microarrays.

Huang WL, Hsu ZJ, Chang TC, Jou R. Rapid and accurate detection of rifampin and isoniazid-resistant Mycobacterium tuberculosis using an oligonucleotide array. Clinical microbiology and infection 2014 Sep; 20 (9): O542-9

Linger, Y., Kukhtin, A., Golova, J., Perov, A., Lambarqui, A., Bryant, L., Rudy, GB, Dionne, K., Fisher, SL, Parrish, N. & Chandler, DP (2014). Simplified microarray system for simultaneously detecting rifampin, isoniazid, ethambutol, and streptomycin resistance markers in Mycobacterium tuberculosis. Journal of clinical microbiology 52, 2100-7

Zhang Z, Li L, Luo F, Cheng P, Wu F, Wu Z, Hou T, Zhong M, Xu J. Rapid and accurate detection of RMP- and INH- resistant Mycobacterium tuberculosis in spinal tuberculosis specimens by CapitalBioTM DNA microarray: A prospective validation study. BMC Infectious Diseases 2012 Nov 14; 12 (1): 303.

Yao C, Zhu T, Li Y, Zhang L, Zhang B, Huang J, Fu W. Detection of rpoB, katG and inhA gene mutations in Mycobacterium tuberculosis clinical isolates from Chongqing as determined by microarray. Clinical microbiology and infection 2010 Nov; 16 (11): 1639-43.

Volokhov DV, Chizhikov VE, Denkin S, Zhang Y. Molecular detection of drug-resistant Mycobacterium tuberculosis with a scanning-frame oligonucleotide microarray. Methods in Molecular Biology 2009; 465: 395-417.

IV.III High Density Oligonucleotide Microchips

Sougakoff W, Rodrigue M, Truffot-Pernot C, Renard M, Durin N, Szpytma M, Vachon R, Troesch A, Jarlier V. Use of a high density DNA probe array for detecting mutations involved in rifampicin resistance in Mycobacterium tuberculosis. Clinical microbiology and infection 2004 Apr; 10 (4): 289-94.

V. Mass spectrometric methods (Matrix-assisted laser desorption ionization-time of flight (mass spectrometry))

Afanasyev M.V., Ikryannikova L.N., Ilyina E.N., Smirnova T.G., Larionova E.E., Kuzmin A.V., Chernousova L.N., Kamaev E.Yu., Skornyakov S .N., Kinsht V.N., Cherednichenko A.G., Govorun V.M. Application of the mini-sequencing reaction followed by MALDI-TOF mass spectrometric analysis to assess resistance to rifampicin and isoniazid of Mycobacterium tuberculosis strains. Tuberculosis and lung diseases. 2007. 84 (7): 27-42

Afanasyev M.V., Borovskaya A.D., Ilyina E.N., Smirnova T.G., Larionova E.E., Kuzmin A.V., Andreevskaya S.N., Chernousova L.N., Govorun V .M. Identification of mutations in the codon 306 of the embB gene for the molecular genetic characteristics of clinical strains of Mycobacterium tuberculosis. Tuberculosis and Pulmonary Diseases 2009. 86 (5): 48-53

Afanas′ev MV, Ikryannikova LN, Il′ina EN, Kuz′min AV, Larionova EE, Smirnova TG, Chernousova LN, Govorun VM. Molecular typing of Mycobacterium tuberculosis circulated in Moscow, Russian Federation. European Journal of Clinical Microbiology and Infectious Diseases 2011 Feb; 30 (2): 181-91.

Ikryannikova LN, Afanas′ev MV, Akopian TA, Il′ina EN, Kuz'min AV, Larionova EE, Smirnova TG, Chernousova LN, Govorun VM. Mass-spectrometry based minisequencing method for the rapid detection of drug resistance in Mycobacterium tuberculosis. Journal of Microbiology Methods 2007 Sep; 70 (3): 395-405.

VI.analysis of conformation polymorphism and heteroduplex analysis using capillary electrophoresis (conformation polymorphism analysis and heteroduplex mobility analysis using temperature gradient capillary electrophoresis)

Krothapalli S, May MK, Hestekin CN. Capillary electrophoresis-single strand conformation polymorphism for the detection of multiple mutations leading to tuberculosis drug resistance. Journal of Microbiology Methods 2012 Oct; 91 (1): 147-54.

Shi R, Otomo K, Yamada H, Tatsumi T, Sugawara I. Temperature-mediated heteroduplex analysis for the detection of drug-resistant gene mutations in clinical isolates of Mycobacterium tuberculosis by denaturing HPLC, SURVEYOR nuclease. Microbes and Infection 2006 Jan; 8 (1): 128-35.

VII. Conjugates of nanoligands with specific DNA probes (nanoprobes for detection of SNPs associated with antibiotic resistance).

Veigas B, Machado D, Perdigão J, Portugal I, Couto I, Viveiros M, Baptista PV. Au-nanoprobes for detection of SNPs associated with antibiotic resistance in Mycobacterium tuberculosis. Nanotechnology 2010 Oct 15; 21 (41): 415101.

Viii. Sequencing of M. tuberculosis gene sequences in which mutations responsible for drug resistance are possible. (DNA sequencing, Next-generation sequencing)

Daum LT, Rodriguez JD, Worthy SA, Ismail NA, Omar SV, Dreyer AW, Fourie PB, Hoosen AA, Chambers JP, Fischer GW. Next-Generation Ion Torrent Sequencing of Drug Resistance Mutations in Mycobacterium tuberculosis Strains. Journal of clinical microbiology 2012 Dec; 50 (12): 3831-7.

Liu CH, Li HM, Lu N, Wang Q, Hu YL, Yang X, Hu YF, Woo PC, Gao GF, Zhu B. Genomic sequence based scanning for drug resistance-associated mutations and evolutionary analysis of multidrug-resistant and extensively drug -resistant Mycobacterium tuberculosis. Journal of Infection 2012 Nov; 65 (5): 412-22.

Si J, Wang Z, Wang Z, Li H. Sequencing-based detection of drug-resistant Mycobacterium tuberculosis in patients with spinal tuberculosis. Archives of Orthopedic and Trauma Surgery 2012 Jul; 132 (7): 941-5.

Feuerriegel S, Oberhauser B, George AG, Dafae F, Richter E, Rüsch-Gerdes S, Niemann S. Sequence analysis for detection of first-line drug resistance in Mycobacterium tuberculosis strains from a high-incidence setting. BMC Microbiology. 2012 May 30; 12:90.

Feuerriegel S, Cox HS, Zarkua N, Karimovich HA, Braker K, Rüsch-Gerdes S, Niemann S. Sequence analyses of just four genes to detect extensively drug-resistant Mycobacterium tuberculosis strains in multidrug-resistant tuberculosis patients undergoing treatment. Antimicrobial Agents and Chemotherapy 2009 Aug; 53 (8): 3353-6

IX. Multiplex ligand-dependent amplification on microspheres. (Multiplex Ligation-dependent Probe Amplification (MLPA) on a Bead Based Array)

Sengstake S, Bablishvili N, Schuitema A, Bzekalava N, Abadia E, de Beer J, Tadumadze N, Akhalaia M, Tuin K, Tukvadze N, Aspindzelashvili R, Bachiyska E, Panaiotov S, Sola C, van Soolingen D, Klatser P, Anthony R, Bergval I. Optimizing multiplex SNP-based data analysis for genotyping of Mycobacterium tuberculosis isolates. BMC Genomics. 2014 Jul 7; 15: 572

Gomgnimbou MK, Hernández-Neuta I, Panaiotov S, Bachiyska E, Palomino JC, Martin A, del Portillo P, Refregier G, Sola C. Tuberculosis-spoligo-rifampin-isoniazid typing: an all-in-one assay technique for surveillance and control of multidrug-resistant tuberculosis on Luminex devices. Journal of Clinical Microbiology 2013 Nov; 51 (11): 3527-34

Bergval, I., Sengstake, S., Brankova, N., Levterova, V., Abadia, E., Tadumaze, N., Bablishvili, N., Akhalaia, M., Tuin, K., Schuitema, A., Panaiotov, S., Bachiyska, E., Kantardjiev, T., de Zwaan, R., Schurch, A., van Soolingen, D., van 't Hoog, A., Cobelens, F., Aspindzelashvili, R., Sola, C., Klatser, P. & Anthony, R. Combined species identification, genotyping, and drug resistance detection of Mycobacterium tuberculosis cultures by MLPA on a bead-based array. PLoS One 2012 7, e43240.

Methods based on real-time PCR (PCR-RV) (I) are most effective for identification, including quantitative, mycobacterium tuberculosis complex in clinical samples. However, these approaches are limitedly applicable for the simultaneous identification of a large (> 100) number of single nucleotide polymorphisms in the genome, which is required when analyzing the determinants of drug resistance and genotyping of the tuberculosis pathogen. An analysis of each mutation requires an individual tube (well of a plate), respectively, while simultaneously determining ~ 100 mutations in one DNA sample, as required when analyzing a patient's DNA for XDR TB, an appropriate number of tubes (wells) will be required, without taking into account the positive control. Taking into account the time of PCR-RV - not less than 1.5 hours, the total time spent on each sample taking into account the DNA extraction (at least 1 hour) will be at least 3 hours. The next DNA sample will also be analyzed in 1.5 hours, etc., thus, per working shift (8 hours) in the presence of only one high-performance DNA amplifier using a 384-well plate, the number of samples analyzed will not exceed 5. The actual needs of the dispensaries are assessed at the level of 30 patients per day. For this reason, PCR-PB based techniques reveal a limited set of mutations and are not applicable for the simultaneous detection of the entire spectrum of mutations in the genome of mycobacterium tuberculosis complex, leading to multiple and wide drug resistance, not to mention simultaneous genotyping.

Cepheid's WHO-recommended Xpert® MTB / RIF (II) replacement cartridge system from Cepheid identifies the 10 most common mutations in the rpoB gene responsible for rifampicin resistance and cannot be used to detect XDR TB and establish clinically important M. tuberculosis genotypes .

The commercial INNO-LiPA Rif.TB (III.I) kit, the basic element of which is a nitrocellulose strip (“strip”) with immobilized specific oligonucleotides, reveals only mutations in the rpoB gene responsible for rifampicin resistance.

GenoType® MTBDRplus and GenoType® MTBDRsl kits (Hain lifescience, Germany) (III.II), also using strips with immobilized probes, respectively reveal 10 genetic determinants of resistance to rifampicin and isoniazid and 11 genetic determinants of resistance to fluoroquinoline, . At the same time, for the analysis of one sample for the detection of MDR and XDR, at least 2 separate tests are required (one for the GenoType® MTBDRplus and GenoType® MTBDRsl systems). The described technologies do not allow to establish the genotype of the causative agent of tuberculosis simultaneously with the analysis of the genetic determinants of drug sensitivity.

The AID TB resistance line probe assay (III.III) kit identifies the genetic determinants of MDR and XDR-tuberculosis, however, it does not allow the simultaneous genotyping of the pathogen.

The TB-Biochip-1 and TB-BIOCHIP-2 kits (IV.I), based on low-density hydrogel oligonucleotide microarrays, reveal, respectively, 48 genetic determinants of rifampicin / isoniazid resistance and 9 genetic determinants of fluoroquinolone resistance. Moreover, for the analysis of one sample, at least 2 separate tests are required (one at a time - with the TB-Biochip-1 and TB-BIOCHIP-2 systems). Even a summary analysis of each sample using the TB-Biochip-1 and TB-BIOCHIP-2 test systems will not reveal TB strains with extensive drug resistance. Moreover, in the analysis of each DNA sample, two consecutive stages of multiplex PCR with mandatory electrophoretic control of PCR products after each stage are required, which complicates the procedure and increases the analysis time.

Alternative methods based on low-density DNA microarrays (IV.II) reveal individual mutations responsible for resistance to rifampicin, isoniazid fluoroquinolones, kanamycin, amikacin, ethambutol, streptomycin, pyrazinamide, but do not provide a comprehensive analysis of the tuberculosis pathogen genome, including identification determinant of resistance and genotyping.

High-density microarrays (IV.III) were developed only for analysis of mutations in the rpoB gene, moreover, judging by the latest trends in the development of molecular genetic tests, this technology did not find consumers in the clinical laboratory diagnosis of tuberculosis.

Mass spectrometric methods (V), analysis of conformational polymorphism and heteroduplex analysis using capillary electrophoresis (VI) and hybridization approaches using nanoligands (VII) provide identification of a limited set of mutations in the M. tuberculosis genome associated with drug resistance, but do not allow for simultaneous genotyping.

Despite the rapid development of new-generation sequencing technologies (VIII), including targeted and genome-wide / metagenome sequencing, transcriptional profiling, etc., these methods remain expensive and are not yet applicable for mass parallel analysis and / or screening of clinical material under flow tests (dozens and hundreds of samples per day) of the clinical diagnostic laboratory (Lohmann, K. & Klein, C. Next Generation Sequencing and the Future of Genetic Diagnosis. Neurotherapeutics 2014. DOI: 10.1007 / s13311-014-0288-8).

Finally, the described method of multiplex ligand-dependent amplification on microspheres (IX), despite the possibility of simultaneous genotyping and identification of determinants of resistance to a wide range of drugs, requires a multi-stage sample preparation procedure (ligation, amplification, two sequential purification of reaction products) and a difficult interpretation of the results, which makes this approach time-consuming and recommended only for laboratories with highly qualified personnel.

Thus, in this area there is an urgent need to develop a method for the detection of tuberculosis pathogen DNA, with the simultaneous establishment of clinically significant genotypes and identification of mutations associated with multidrug and wide drug resistance, which would be favorably distinguished from the prior art solutions by the ease of analysis, high specificity and informativeness in relation to the number of determined determinants of stability, as well as low cost.

Disclosure of invention

As a result of extensive scientific research and analysis of the NCBI nucleotide sequence database http://www.ncbi.nlm.nih.gov/genome/genomes/166?subset=, Welcome Trust Sanger institute http://www.sanger.ac. uk / resources / downloads / bacteria / mycobacterium.html, TBDReaMDB (www.tbdreamdb.com), the authors of the present invention found that the task of developing a method for detecting tuberculosis pathogen DNA, with the simultaneous establishment of the genotype and identification of mutations associated with multidrug and extensive drug resistance can be successfully solved by using oligonucleotide micro types (biochips) containing oligonucleotide probes, the sequences of which are specific to the fragment of the insertion element IS6110, characteristic of the causative agent of tuberculosis, single nucleotide polymorphisms, which establish belonging to the genotypes Beijing, Haarlem, LAM, Ural, as well as mutant variants of the rpoA, kGG, inh genes ahpC, gyrA, gyrB, rrs, eis, embB, which are drug resistance markers of M. tuberculosis.

The method claimed in the present invention compares favorably with the methods known from the prior art by the ability to identify the pathogen directly in the clinical material with the simultaneous establishment of the Beijing, Beijing B0 / W148, Haarlem, LAM, Ural genotypes and the determination of 120 mutations determining resistance to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin, capreomycin, ethambutol. The method does not require expensive equipment and highly qualified personnel. The data obtained using the claimed method can be used to personify the selection of effective anti-TB drugs, to identify clinically significant especially dangerous forms of the pathogen (for example, the Beijing family) and epidemiological genotyping.

In its first aspect, this invention provides a method for detecting tuberculosis pathogen DNA while simultaneously determining its genotype and determining the genetic determinants of resistance to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin, capreomycin, ethambutol. The method is based on multiplex PCR to obtain fluorescently labeled fragments of the mycobacterial genome, followed by hybridization of these fragments on an oligonucleotide microchip containing a set of differentiating oligonucleotides.

The method comprises the following steps:

a) multiplex amplification of rpoB, katG, inhA, ahpC, gyrA, gyrB, rrs, eis, embB genes, mobile element IS6110, fragments of the mycobacterial genome containing genotype-specific single nucleotide polymorphisms Rv0557, Rv0129c, Rv1811, Rv1829, Rv2629, Rv2629, Rv2629, Rv2629, Rv2629, labeling using the genomic DNA of mycobacterium tuberculosis complex as a template and a set of primers, the sequences of which are represented by SEQ ID NO: 1-36, to obtain fluorescently labeled fragments of the mycobacterial genome.

b) providing an oligonucleotide microchip for identifying the DNA of the Mycobacterium tuberculosis complex with the simultaneous establishment of the Beijing, Beijing B0, LAM, Haarlem, Ural genotypes and the determination of mutations responsible for the resistance of the tuberculosis pathogen to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikamycin, capamicin, amicacolcin, capamicin, amikacin, a substrate containing many discrete elements, in each of which a unique oligonucleotide probe is immobilized, having a sequence complementary to lnosti single-chain fragment obtained in step (a) and selected from the group consisting of: a) sequences corresponding to a fragment of IS6110 mobile element; b) the corresponding sequence of the fragment, including the Rv0557 polymorphism characteristic of the Euro-American line; c) the corresponding sequence of the fragment, including the Rv0557 polymorphism characteristic of the Haarlem genotype, with established affiliation with the Euro-American line; d) the corresponding sequence of the fragment, including the Rv0129c polymorphism characteristic of the LAM genotype, with established affiliation with the Euro-American line; e) the corresponding sequence of the fragment, including the Rv1811 polymorphism characteristic of the Ural genotype, with established affiliation with the Euro-American line; f) the corresponding sequence of the fragment, including the Rv2629 polymorphism characteristic of the Beijing family, in the absence of belonging to the Euro-American line; g) the corresponding sequence of the fragment, including the Rv0118c polymorphism in the oxcA gene, characteristic of the Beijing B0 / W148 genotype with the installed Beijing family; h) the corresponding sequences of the wild-type rpoB gene fragment; i) the corresponding sequence of the fragment of the mutant variant of the rpoB gene, leading to the resistance of microorganisms to rifampicin; i) the corresponding sequences of the wild-type katG gene fragment; k) the corresponding sequence of the fragment of the mutant variant of the katG gene, leading to the resistance of microorganisms to isoniazid; k) the corresponding sequences of the wild-type inhA gene fragment; m) the corresponding sequence of the fragment of the mutant variant of the inhA gene, leading to the resistance of microorganisms to isoniazid; m) the corresponding sequences of the wild-type ahpC gene fragment; o) the corresponding sequence of the fragment of the mutant variant of the ahpC gene, leading to the resistance of microorganisms to isoniazid; o) the corresponding sequences of the wild-type gyrA gene fragment; p) the corresponding sequence of the fragment of the mutant variant of the gyrA gene, leading to the resistance of microorganisms to fluoroquinolones; c) the corresponding sequences of the wild-type gyrB gene fragment; r) the corresponding sequence of the fragment of the mutant variant of the gyrB gene, leading to the resistance of microorganisms to fluoroquinolones; s) the corresponding sequence of the fragment of the wild-type eis gene; f) the corresponding sequence of the fragment of the mutant variant of the eis gene, leading to the resistance of microorganisms to kanamycin, amikacin and capreomycin; x) the corresponding sequences of the wild-type rrs gene fragment; c) the corresponding sequence of the fragment of the mutant variant of the rrs gene, leading to the resistance of microorganisms to kanamycin, amikacin and capreomycin; h) the corresponding sequences of the wild-type embB gene fragment; sh) the corresponding sequence of the fragment of the mutant variant of the embB gene, leading to the resistance of microorganisms to ethambutol; wherein the sequences of immobilized oligonucleotide probes are represented by SEQ ID NO: 37-229;

(c) - hybridization of the amplified fluorescently-labeled products obtained in stage (a) on an oligonucleotide microchip with the formation of duplexes with immobilized probes under conditions providing a resolution of one nucleotide between perfect and imperfect duplexes formed as a result of hybridization;

(d) - recording the results of hybridization on an oligonucleotide microchip, carried out in stage (c) using a portable fluorescence analyzer and software, which allows the use of software processing of signal intensities with subsequent interpretation of the results, which is carried out in two stages: at the first stage, the signals are analyzed a microchip cell containing an oligonucleotide probe specific for a fragment of the mobile element IS6110, characteristic of the mycobacterium tuberculosis complex, with the aim of aruzheniya DNA of Mycobacterium tuberculosis; in the presence of a signal in this cell, i.e. in the case of detection of mycobacterial DNA, in the second stage, microarray elements containing genotype-specific oligonucleotide probes that study the polymorphisms Rv0557, Rv0129c, Rv1811, Rv2629, Rv0118c, and elements in which oligonucleotide probes are detected for inhA, ahpC, gyrA, gyrB, rrs, eis, embB, thereby establishing the genotype of the causative agent of tuberculosis and determining the genetic determinants of resistance to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin, capreomycin, ethambutol.

In one of its embodiments, the method is characterized in that the amplification of fragments of the mycobacterial genome is carried out using DNA isolated from the material of a clinical sample (sputum, exudate, bronchoalveolar lavage, blood) or a pre-grown culture of the microorganism.

In a further embodiment, the method is characterized in that in step (a) c, fluorescently labeled deoxyuridine triphosphate is used to fluorescently label the resulting PCR fragments.

In yet another embodiment, the method is characterized in that in step (d) in the second step of interpreting the results in the analysis of microarray elements containing genotype-specific oligonucleotide probes, if the wild-type Rv0557_321 polymorphism is established, the strain is referred to the Euro-American line, with further identification of the Haarlem genotype (by Rv0557_455 polymorphism), or the LAM genotype (by Rv0129c polymorphism), or Ural (by Rv1811 polymorphism); when another variant of Rv0557_321 polymorphism is detected, the strain is assigned to a different, non-Euro-American line, with further identification of the Beijing family (by Rv2629 polymorphism), and determination of the B0 / W148 genotype for the Beijing family (by Rv0118 polymorphism).

In yet another embodiment, the method is characterized in step (d) in the second step of interpreting the results; groups of elements containing oligonucleotide probes specific to one of the rpob, katG, inhA, ahpC, gyrA, gyrB, rrs, eis, tmbB genes are analyzed; each group includes an element containing a wild-type probe, and elements containing probes corresponding to mutant gene variants. If the maximum signal in the group is registered in an element containing a wild-type probe, then it is believed that the DNA sample of the tuberculosis causative agent does not have mutations by the given amino acid / nucleotide position of this gene. If the maximum signal in the group is registered in the element containing the probe, the sequence of which is specific for the mutant variant of one of the genes, then it is believed that for a given amino acid / nucleotide position of this gene, the studied DNA sample of the tuberculosis pathogen has an amino acid / nucleotide substitution, which is a genetic determinant of resistance.

Finally, in yet another embodiment, the method may further include confirming the clinical diagnosis of "tuberculosis", personalizing the selection of effective chemotherapeutic agents of the first and second row, establishing clinically significant genotypes and epidemiological genotyping based on the interpretation of the results.

Short list of figures

For a clearer understanding of the essence of the claimed invention, as well as to demonstrate its characteristic features and advantages, the following is a detailed description of the invention with reference to the figures of the drawings, in which:

FIG. 1. represents the layout of discriminating oligonucleotides on a biochip.

FIG. 2. The fluorescence pattern of hybridization of the biochip and the interpretation results for DNA analysis of the laboratory strain M. tuberculosis H37Rv, sensitive to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin / capreomycin, ethambutol.

FIG. 3. Fluorescence picture of hybridization of the biochip and interpretation results in the analysis of DNA isolated from a clinical sample containing a strain of M. tuberculosis of the Ural genotype, which is resistant to isoniazid.

FIG. 4. Fluorescence pattern of hybridization of the biochip and interpretation results in the analysis of DNA isolated from a clinical sample containing the M. tuberculosis strain of the Beijing B0 / W148 genotype, which is resistant to rifampicin, isoniazid, ethambutol.

FIG. 5. Fluorescence pattern of biochip hybridization and interpretation results when analyzing DNA isolated from a clinical sample containing a M. tuberculosis strain of the Beijing genotype, resistant to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin / capreomycin, ethambutol.

FIG. 6. Fluorescence picture of hybridization of the biochip and interpretation results when analyzing DNA isolated from a clinical sample containing a strain of M. tuberculosis of the LAM genotype, which is resistant to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin / capreomycin, ethambutol.

FIG. 7. The fluorescence pattern of hybridization of the biochip and interpretation results in the analysis of DNA isolated from a clinical sample that does not contain mycobacterium tuberculosis complex.

The implementation of the invention

An object of the present invention is to provide a method for detecting tuberculosis pathogen DNA with simultaneous determination of its genotype and determination of the genetic determinants of multiple and broad drug resistance using an oligonucleotide microchip.

The claimed method proposes the use of multiplex amplification and fluorescence labeling of fragments of the mycobacterial genome to obtain fluorescently-labeled PCR products, while genomic DNA isolated from a clinical sample, such as, for example, sputum, bronchial lavage, can be used as a matrix; bronchoalveolar swabs, material obtained by bronchoscopy, transtracheal and intrapulmonary biopsy; bronchi aspirate; laryngeal smears; exudates; swabs from thoracic wounds, as well as mycobacterial cell cultures obtained with liquid and solid media. The claimed method also involves the use of an original oligonucleotide microchip with immobilized specific oligonucleotide probes, hybridization procedures, registration and interpretation of the results, as well as sets of primers and oligonucleotide probes used to implement the method.

Schematic diagram of the analysis of mycobacterial DNA with the aim of identifying the mycobacterium tuberculosis complex, establishing the pathogen genotype and determining the genetic determinants of resistance to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin, capreomycin, ethambutol.

A clinical sample is subjected to decontamination and cell lysis to ensure access to genomic DNA. One suitable method is liquefying under alkaline conditions in the presence of N-acetyl-L-cysteine and boiling with detergent to provide access to DNA and decontamination of the sample (Kent, PT, and GP Kubica. Public health mycobacteriology. A guide for level III laboratory. 1985 Centers for Disease Control and Prevention, Atlanta, GA). Other methods known to those skilled in the art can also be used for these purposes, such as cell disruption by ultrasound (Padilla E, Gonzalez V, Manterola JM, et al. Evaluation of two different cell lysis methods for releasing mycobacterial nucleic acids in the INNO-LiPA mycobacteria test. Diagn Microbiol Infect Dis. 2003 May; 46 (1): 19-23), lysis with guanidine thiocyanate - sarcosine (Chakravorty S, Tyagi JS. Novel use of guanidinium isothiocyanate in the isolation of Mycobacterium tuberculosis DNA from clinical material. FEMS Microbiology Letters 2001 Nov 27; 205 (1): 113-7.) etc. Genomic DNA purification after lysis can be performed using automated robotic stations, for example, Freedom EVO® Clinical (Tecan Group Ltd., Germany), or commercial kits, such as, for example, PROBA-NK (NPO DNA- Technology ", Registration certificate of Roszdravnadzor No. FSR 2008/02938)," Reagent in test tubes for DNA extraction from biological samples for the subsequent analysis by polymerase chain reaction (DNA-EXPRESS) "(LLC NPF Litekh, Registration certificate of Roszdravnadzor FSR 2007 / 00362), reagent kit A RNA / DNA extraction from clinical material "ribo-prep" TU 9398-071-01897593-2008 (FBUN Central Research Institute of Epidemiology, registration certificate Roszdravnadzor FSS 2008/03147).

The resulting preparation of mycobacterial genomic DNA is used as a template in multiplex PCR. During amplification, fragments of rpoB, katG, inhA, ahpC, gyrA, gyrB, rrs, eis, embB genes, mobile element IS6110, fragments of the mycobacterial genome containing genotype-specific single nucleotide polymorphisms Rv0557, Rv0129c, Rv1811, Rv26, are produced.

Primers for conducting multiplex PCR are selected so that they flank the region of the gene or regulatory region where the most common mutations are found, leading to the resistance of the microorganism to chemotherapeutic agents, or genotype-specific single nucleotide polymorphisms .. Using specialized software, for example, Oligo v. 6.3 (Molecular Biology Insights Inc., USA) or Fast PCR (http://www.biocenter.helsinki.fi/bi/Programs/fastpcr.htm) or other commercially available programs, or programs freely available on the Internet, are calculated the melting temperatures of the primers and, varying their length, ensure that the scatter in the annealing temperatures of the primers inside the kit does not exceed 3-4 ° C. When selecting primers, sequences that are capable of forming secondary structures such as hairpins with high melting points are avoided. When choosing primers for multiplex PCR, sequences that form duplexes of more than three to five nucleotides between themselves are avoided. Each selected primer should have unique specificity with respect to the analyzed region of the nucleic acid sequence of the genome of mycobacterium tuberculosis complex. The specificity of the primers is checked using software that uses the search in the nucleotide sequence databases using the BLAST algorithm (for example, www.ncbi.nlm.nih.gov/BLAST).

To ensure effective amplification with the simultaneous introduction of a fluorescent label in all of the above segments of the mycobacterial genome in a single reaction volume, primers of the following design are used. The sequence of each primer added to the reaction mixture consists of two parts - 3′-specific, i.e. complementary sequence of a fragment of the genome of M. tuberculosis, and 5′-universal (adapter), which was different for direct and reverse primers. In addition to such primers containing both specific and adapter sequences, two primers are added to the reaction mixture, the sequences of which are complementary to the sequences of the adapter portion of the composite primers (SEQ ID NO: 1, 2, Table 1). These adapter primers are present in the reaction mixture in various concentrations in order to produce predominantly single-stranded fragments whose sequences are complementary to oligonucleotide sequences immobilized on a biochip.

The calculated melting points of the specific and adapter sequences are chosen equal to 70 ° C and 55 ° C, respectively, and the amplification profile includes two stages of 30 cycles each, with annealing temperatures of 64 ° C in the first and 50 ° C in the second stage.

Thus, in the course of PCR in a single reaction volume in the first stage due to hybridization and elongation of composite primers using genomic DNA as a template, double-stranded PCR products containing sequences specific to adapter primers at the ends are produced, and then, during the second stages obtained PCR products serve as a matrix for the production of single-chain fragments using adapter primers with a lower annealing temperature.

The fluorescent labeling of the studied fragments of the mycobacterial genome is carried out by adding a fluorescent substrate, a conjugate of deoxyuridine triphosphate and dye of the indodicarbocyanine series, with an excitation wavelength of (640 ± 5) nm and a fluorescence wavelength of (665O12 ± 13 nm) (665O12 nm) in the PCR mixture. Indicyanine dyes and the derivatives resulting for analysing biological micromolecules). During PCR, this substrate is inserted by Taq DNA polymerase into the growing DNA strand, providing fluorescence-labeled PCR products that are further analyzed by biochip hybridization (Alexandrova LA, Jasko MV, Belobritskaya EE, Chudinov AV, Mityaeva ON, Nasedkina TV , Zasedatelev AS, Kukhanova MK. New triphosphate conjugates bearing reporter groups: labeling of DNA fragments for microarray analysis. Bioconjugate Chemistry 2007 18 (3): 886-93).

When choosing discriminating oligonucleotides for immobilization on a biochip, taking into account the size and complexity of the analyzed sequence and, in particular, the presence of repeats and extended homopolymer sequences, the length of the discriminating oligonucleotides, which ensures their specificity with respect to the analyzed sequence, is determined. For each position for which mutations or single nucleotide polymorphism are known, a set of specific discriminating oligonucleotides is selected that is capable of detecting known substitution variants. Using software such as Oligo v. 6.3 (Molecular Biology Insights Inc., USA), calculate the melting temperature of oligonucleotides and, varying their length, ensure that the dispersion of the melting temperature of oligonucleotides is not more than 2-3 ° C. Avoid such oligonucleotides that are capable of forming secondary structures such as hairpins with high melting points. The position of the determined variable nucleotides and other nucleotide rearrangements is selected, if possible, no further than 1-4 nucleotides from the middle of the corresponding discriminating oligonucleotide.

Discriminating oligonucleotides are immobilized in gel elements, which are applied to a glass slide format substrate in the form of droplets with a diameter of 50 to 100 μm with a period of 50-100 μm, without the use of special devices, for example, quartz masks. The substrate material used is polymers (polypropylene, polyethylene, polybutylene terephthalate, polymethacrylate, polycarbonate, polystyrene), or glass. Under the action of ultraviolet radiation, joint polymerization of oligonucleotides with the main components of the gel occurs. As a result of this reaction, the capture molecule is covalently attached to the monomers of the growing polymer chains and uniformly distributed throughout the volume of each gel cell (Rubina AY, Pan 'kov SV , Dementieva EI et al Hydrogel drop microchips with immobilized DNA:. Properties and methods for large-scale production. Analytical Biochemistry 2004; 325: 92-106).

PCR products obtained at the PCR stage are hybridized on a differentiating biochip with immobilized oligonucleotides complementary to the sequences studied for the presence of gene mutations, a fragment of the insertion element IS6110, fragments containing a genotype-specific single nucleotide polymorphism. Hybridization is carried out in a solution containing a buffer component to maintain pH, a salt to create ionic strength and a chaotropic (destabilizing hydrogen bond) agent in a sealed hybridization chamber at a temperature depending on the melting temperature of discriminating oligonucleotides immobilized on a microchip. As a hydrogen destabilizing agent, for example, guanidine thiocyanate, urea or formamide can be used. The choice of the optimum hybridization temperature is carried out taking into account the convenience of practical application of the system. The discriminatory oligonucleotides of the present invention have a melting point in the range of 42 to 44 ° C, which allows hybridization at 37 ° C using a chaotropic agent. The temperature of 37 ° C is convenient because most clinical laboratories are equipped with thermostats that maintain this temperature.

The analyzed DNA fragments form perfect hybridization duplexes only with the corresponding (fully complementary) oligonucleotides. With all other oligonucleotides, the studied DNA fragments give an imperfect duplex. Discrimination of perfect and imperfect duplexes is performed by comparing the fluorescence intensities of the cells in which the duplexes formed. The signal intensity in the cell in which the perfect hybridization duplex (I _owls ) was formed is higher than in that where the imperfect duplex (I _non-waves ) was formed. Hybridization under optimal conditions (temperature, selected concentration of a chaotropic agent and the ionic strength of the hybridization buffer) allows one to achieve a ratio of I _ow / I _nons ≥ 2 between two cells containing probes belonging to the same group and differing by one nucleotide.

Using the arrangement of oligonucleotides on the biochip (Fig. 1), the presence of tuberculosis pathogen DNA is determined based on the presence of a signal in a cell containing an IS probe specific for fragment IS6110, which is characteristic only of mycobacterium tuberculosis complex. The interpretation of the result in this group is based on the ratio of the signal _{IS IS} in the _IS cell with the average signal I _<0> in the comparison cells '0' that do not contain immobilized oligonucleotides. If the ratio I _IS / I _<0> ≥ 4 is fulfilled for this group, then it is believed that DNA belonging to the mycobacterium tuberculosis complex is found in the analyzed sample.

The establishment of the pathogen genotype is based on a comparison of signals in groups of cells containing oligonucleotides whose sequences are complementary to fragments containing genotype-specific single nucleotide polymorphisms Rv0557, Rv0129c, Rv1811, Rv2629, Rv0118c. Cells t321, 321c, 455, 455c are used to identify Rv0557 polymorphism; Rv0129 - g309, 309a; Rv1811 - g12, 12a; Rv2629 - a191, 191c; Rv0118c - g757, 757t (Fig. 1). The signal intensities within these groups of cells are also compared if the maximum signal in one of the cells exceeds the signals in others by more than 2 times, i.e. the ratio of I _owls / I _nesov ≥ 2 is satisfied between cells belonging to one group, make the following conclusions:

- if the wild-type is established by the Rv0557_321 polymorphism, the strain is assigned to the Euro-American line, with further identification of the Haarlem genotype (by the Rv0557_455G> C polymorphism), or the LAM genotype (by the Rv0129c_309G> A polymorphism), or Ural (by the Rv1829c_309G> A polymorphism R11> G11>) ;

- when the replacement T> C of the Rv0557_321 polymorphism is detected, the strain is assigned to a different, non-Euro-American line, and if the Rv2629_191A> C polymorphism is present, the Beijing family is determined.

- if the Beijing family is detected, cells g757, 757t are analyzed. When the replacement Rv0118c G> T is detected in the oxcA gene, the B0 / W148 genotype for the Beijing family is established.

In each group of cells containing oligonucleotides specific to one of the rpob, katG, inhA, ahpC, gyrA, gyrB, rrs, eis, embB genes, there is a cell containing a wild-type probe (circled by the thick line in Fig. 1) and cells containing probes for possible mutations. For each group of cells, the maximum signal is recorded that exceeds the others by more than 2 times (the ratio of I _owls / I _incons ≥ 2 is checked). If the maximum signal is registered in a cell corresponding to DNA without mutations (i.e., belonging to a microorganism that is sensitive to a drug), then it is believed that the studied sample of mutations does not have a mutation at this amino acid / nucleotide position (group of cells). If the maximum signal is registered in a cell corresponding to DNA with a mutation (mutations) (i.e., belonging to a drug-resistant microorganism), then it is considered that for a given amino acid / nucleotide position (group of cells) the sample under study has an amino acid / nucleotide substitution, leading to resistance. The studied DNA sample is recognized as belonging to a sensitive strain of mycobacteria if, for each variable amino acid / nucleotide position (group of cells), the sample is characterized as sensitive. The studied DNA sample is recognized as belonging to a resistant strain of mycobacteria if at least one variable amino acid / nucleotide position (group of cells) is characterized as having a mutation leading to resistance. For such samples, the type of drug to which resistance is detected is additionally determined by establishing the group by which the sample is assigned to the resistant type.

This algorithm can be implemented in software that allows automatic recording and interpretation of results by capturing a fluorescence picture of hybridization, calculating the signal in each cell, comparing signals within groups and reporting on the presence of tuberculosis pathogen DNA in the analyzed sample, its genotype and absence / the presence of the determinant of resistance to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin, capreomycin, ethambutol.

The results can be used to confirm the clinical diagnosis of tuberculosis (if there is an appropriate clinical picture and the presence of the tuberculosis complex mycobacteria in the DNA sample), for personalized prescribing of a spectrum of effective chemotherapeutic agents, for which the absence of genetic resistance determinants was shown, to identify clinically significant pathogen genotypes for example, Beijing and Beijing B0, characterized by increased virulence and transmissibility, and, finally, for the epidemic biological genotyping, including the determination of the most common genotypes in the Russian Federation, Haarlem, LAM, Ural, Beijing, Beijing B0.

The invention will now be illustrated by examples, which are intended to provide a better understanding of the essence of the claimed invention, but should not be construed as limiting the invention.

Examples

Example 1. Treatment of a clinical sample and DNA isolation. Conducting multiplex PCR and hybridization on an oligonucleotide microchip. Registration and interpretation of hybridization results.

Clinical sample processing and DNA isolation.

1. A clinical sample (sputum, exudate, flush, bronchoalveolar lavage, blood, etc.) was mixed in a 1: 1 ratio by volume with a freshly prepared 0.5% solution of N-acetyl-L-cysteine (NALC) in 2% NaOH . The sample was thoroughly mixed on a vortex and kept at room temperature for 20 minutes. A pH of 6.8 phosphate buffer was added to the sample in a ratio of 1: 5 by volume and centrifuged for 30 min at 3000 rpm. When using cerebrospinal fluid, preliminary centrifugation was performed for 10 min at 10,000 rpm. During blood analysis, the lymphocyte fraction was preliminarily isolated according to the generally accepted method using ficoll. Further processing of the samples was carried out identically.

2. Cell pellet was suspended in 1.5 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA), pH 8.0, and precipitated at 3000 rpm for 30 minutes. The washing was repeated once more.

3. To the resulting precipitate was added 30 μl of TE buffer, pH 8.0, containing 1% (vol./about.) Triton X-100 and kept in a dry thermostat at 95 ° C for 30 minutes.

4. The sample was centrifuged at 10,000 rpm for 10 minutes.

5. Further DNA purification was performed using the PROBA-NK kit (NPO DNA-Technology LLC) according to the manufacturer's instructions. The resulting DNA solution in a volume of 50 μl is suitable for multiplex PCR.

Carrying out multiplex PCR.

For each analyzed sample, 2 sterile tubes with a capacity of 0.2 ml were prepared for PCR, labeling them as "N ₁ " and "N ₂ ", where N is the number of the analyzed sample.

The composition of the PCR mixture in vitro N ₁

- 1X PCR buffer for HS Taq DNA polymerase (ZAO Evrogen, Russia ");

- 200 μM of each dATP, dCTP, dGTP, dUTP (Eurogen);

- 1 μm of a fluorescent substrate IMD515-dUTP (LLC BIOCHIP-IMB, Russia)

- A mixture of primers PR-1. The sequence of the primers and their concentration in a mixture of PR-1 are presented in Table 1.

- 5 units HS Taq DNA polymerase (ZAO Eurogen, Russia ");

The composition of the PCR mixture in vitro N ₂

- 1X PCR buffer for HS Taq DNA polymerase (ZAO Evrogen, Russia ");

- 200 μM of each dATP, dCTP, dGTP, dUTP (Eurogen);

- 1 μm of a fluorescent substrate IMD515-dUTP (LLC BIOCHIP-IMB, Russia)

- A mixture of primers PR-2. The sequence of the primers and their concentration in a mixture of PR-2 are presented in Table 1.

- 5 units HS Taq DNA polymerase (ZAO Eurogen, Russia ");

In 30 μl of PCR mixtures N ₁ and N _2, 3 μl of the DNA solution obtained in step 5 of the clinical sample processing and DNA isolation procedure was added.

Table 1 The list of sequences and concentrations of primers used in the reaction mixtures of the multiplex PCR stage of the proposed method Seq ID No Primer Name 5'-3 'sequence Gene / target Reaction mixture The concentration in the reaction mixture, µmol one lm_fa CTCTGCGAGCATAATG adapter PR-1, PR-2 one hundred 2 lm_ra GGCTGTACGCTGTC adapter PR-1, PR-2 2000 3 rpoB..F CTCTGCGAGCATAATGAGTGAGACGGGTGCACGTCGCGGACCT rpoB PR-1 fifty four rpoB..R GGCTGTACGCTGTCCGTGGAGGCGATCACACCGCAGACGTTG rpoB PR-1 fifty 5 ahpC..F CTCTGCGAGCATAATGATGGTGTGATATATCACCTTTGCCTGACAGC ahpC PR-1 fifty 6 ahpC..R GGCTGTACGCTGTCTGACTCTCCTCATCATCAAAGCGGACA ahpC PR-1 fifty 7 katG..F CTCTGCGAGCATAATGTGGAAGAGCTCGTATGGCACCGGAACC katG PR-1 40 8 katG..R GGCTGTACGCTGTCGCTCTCCGTCAGCTCCCACTCGTAGCC katG PR-1 40 9 inhA..F CTCTGCGAGCATAATGCCGGAAATCGCAGCCACGTTACGC inhA PR-1 thirty 10 inhA..R GGCTGTACGCTGTCGGTAACCAGGACTGAACGGGATACGAA inhA PR-1 thirty eleven IS6110..F CTCTGCGAGCATAATGAGGATGGGGTCATGTCAGGTGGTTCATCGA IS6110 PR-1 thirty 12 IS6110..R GGCTGTACGCTGTCGCGAAGAAAGAGGACGCGGTCTTTAAAATC IS6110 PR-1 thirty 13 embB (296) F CTCTGCGAGCATAATGCCCTGACCGACGCCGTGGTGATATTCG embB PR-1 thirty fourteen embB (328) R GGCTGTACGCTGTCCGCCAGCAGGTTGTAATACCAGCCGAAGG embB PR-1 thirty fifteen embB (354) F CTCTGCGAGCATAATGCGACGCCAGTCTGTGGATGCGCCTG embB PR-2 fifty 16 embB (406) R GGCTGTACGCTGTCGGACCGCTCGATCAGCACATAGGTGACCA embB PR-2 fifty 17 embB (497) F CTCTGCGAGCATAATGCCGTCATCCTGACCGTGGTGTTCGCC embB PR-2 fifty eighteen embB (497) R GGCTGTACGCTGTCCGCGAACCCTGGTGGCTTCCAACAC embB PR-2 fifty 19 gyrA..F CTCTGCGAGCATAATGGGAGGTGCGCGACGGGCTCAAGC gyrA PR-1 one hundred twenty gyrA..R GGCTGTACGCTGTCCCAGCGGGGTAGCGCAGCGACCA gyrA PR-1 one hundred 21 gyrB..F CTCTGCGAGCATAATGCAAGGCTGTGTCCTCGGCGCAAGCC gyrB PR-1 fifty 22 gyrB..R GGCTGTACGCTGTCCGGTGCCCAGCGCCGTGATGAT gyrB PR-1 fifty 23 rrs..F CTCTGCGAGCATAATGTCTCAGTTCGGATCGGGGTCTGCAACTCG rrs PR-1 one hundred 24 rrs..R GGCTGTACGCTGTCCCAGTTGGGGCGTTTTCGTGGTGCTCC rrs PR-1 one hundred 25 eis..F CTCTGCGAGCATAATGGCGAAATTCGTCGCTGATTCTCGCAGTGGC eis PR-1 fifty 26 eis..R GGCTGTACGCTGTCCCCGGCCAGTCGTCCTCGGTCG eis PR-1 fifty 27 Rv0129..F CTCTGCGAGCATAATGGCGGTCTACCTGCTCGACGGTCTGCG Rv0129c PR-2 fifty 28 Rv0129..R GGCTGTACGCTGTCGCCGCCCACGGGCATGATCACC Rv0129c PR-2 fifty 29th Rv0557..F CTCTGCGAGCATAATGCCGGATGTCGTGCATCTGGCGTCGC Rv0557 PR-2 fifty thirty Rv0557..R GGCTGTACGCTGTCGGACGGCCCCAGAGTGCGGTCAG Rv0557 PR-2 fifty 31 Rv1811..F CTCTGCGAGCATAATGGCTGCCGTGGCCGTCTTGCGATCG Rv1811 PR-2 fifty 32 Rv1811..R GGCTGTACGCTGTCGCAACCCACTCCCCGACGGCCAGCC Rv1811 PR-2 fifty 33 Rv2629..F CTCTGCGAGCATAATGGCCGCGACGCGAAGCAGGAGCTC Rv2629 PR-2 fifty 34 Rv2629..R GGCTGTACGCTGTCAGATGCTCGTTGACCAGTACTTGCTCGCC Rv2629 PR-2 fifty 35 oxcA..F CTCTGCGAGCATAATGGCCGGCACCGGAGGCGATTGATCG oxcA / Rv0118c PR-2 fifty 36 oxcA..R GGCTGTACGCTGTCGCGGGTGTGAGTCGGGCAGCAGC oxcA / Rv0118c PR-2 fifty

Multiplex PCR was performed on a S1000 programmable thermostat (Bio-Rad, USA) according to the temperature regime shown in Table 2.

table 2 Temperature-time regime of the PCR stage Program step Temperature Incubation time The number of cycles one + 95 ° C (preliminary DNA denaturation) 4 min one 2 + 95 ° C (DNA denaturation) 30 s 40 + 64 ° C (annealing of primers) 30 s + 72 ° С (completion of primers) 30 s 3 + 95 ° C (DNA denaturation) 30 s 40 + 50 ° С (annealing of primers) 30 s + 72 ° С (completion of primers) 30 s four + 72 ° C (final incubation) 5 minutes one

Hybridization and washing on a biochip.

7.5 μl of hybridization buffer (1.5 M guanidine thiocyanate, 75 mM HEPES pH 7.5, 7.5 mM EDTA) was added to a 0.5 ml tube. Contributed 17.5 μl of the reaction mixture from tube No. ₁ , then 5 μl from a tube with index N ₂ after the PCR stage. The resulting mixture was vortexed, drops were collected by centrifugation for 10 s at 1000 g.

30 μl of the obtained hybridization mixture was introduced into one of the openings of the biochip reaction chamber.

Hybridization was carried out at 37 ° C for 6-12 hours. After hybridization, the biochip was washed three times with distilled water at 37 ° C, the hybridization chamber was removed and the biochip was dried in an air stream.

Registration and interpretation of hybridization results.

The fluorescence image of the microchip was recorded on a universal hardware-software complex (UAPK) (BIOCHIP-IMB LLC, Russia) for the analysis of biological microchips using the specialized ImageWare® software (BIOCHIP-IMB LLC).

Interpretation of the results was carried out according to the above algorithm, implemented in the software module for the analysis of fluorescence images of Imageware® microarrays.

Example 2. An oligonucleotide microchip for detecting the DNA of the tuberculosis pathogen with the simultaneous establishment of the pathogen genotype and determination of the genetic determinants of resistance to rifampicin, isoniazid, fluoroquinolones, aminglicosides, capreomycin, ethambutol.

The oligonucleotides were synthesized using an ABI-394 DNA / RNA synthesizer (Applied Biosystems, USA) using the standard phosphoramidite method and purified by reverse phase HPLC (Gilson complex, France). Oligonucleotides for immobilization in the elements of the microchip contained a spacer with a free amino group introduced during the synthesis using 5′-Amino-Modifier C6 (Glen Research, USA).

The oligonucleotides were immobilized on a biochip substrate in a glass slide format as described previously (Pan'kov SV, Chechetkin VR, Somova OG, Antonova OV, Moiseeva OV, Prokopenko DV, Yurasov RA, Gryadunov DA, Chudinov AV. Kinetic effects on signal normalization in oligonucleotide microchips with labeled immobilized probes. Journal of Biomolecular Structure and Dynamics. 2009 27 (2): 235-44).

A biochip diagram is shown in FIG. 1. contained 203 gel elements, including 193 cells with immobilized oligonucleotides, 7 empty gel cells with an index of '0', acting as a negative control and necessary to calculate the background fluorescent signal, and 3 cells with an index of 'M' containing a covalently bound fluorescent dye and used to automatically calculate the fluorescence intensity of biochip cells after hybridization.

The biochip allows you to identify the DNA of Mycobacterium tuberculosis complex (oligonucleotide in the IS cell), establish affiliation with the Euro-American line and determine the genotypes of Haarlem, LAM, Ural, Beijing, Beijing B0, and identify a total of 120 genetic determinants of drug resistance, including:

- 28 mutations in the rpoB gene responsible for rifampicin resistance;

- 11 mutations in the katG gene, 5 mutations in the inhA gene, 5 mutations in the ahpC gene, leading to isoniazid resistance;

- 15 mutations in the gyrA gene, 23 mutations in the gyrB gene, responsible for resistance to fluoroquinolones;

- 4 mutations in the rrs gene, 6 mutations in the eis gene, leading to resistance to kanamycin, amikacin and capreomycin;

- 23 mutations in the embB gene responsible for resistance to ethambutol.

Cells containing oligonucleotides for identifying the determinants of drug resistance are combined into groups corresponding to variable amino acid residues or nucleotides in the regulatory regions of genes. In each group, there is an element containing an oligonucleotide capable of forming a perfect hybridization duplex with DNA without mutations (i.e., wild-type (WT) DNA) at positions corresponding to the following amino acid residues or nucleotides in the regulatory regions of genes (such the cells are surrounded by a thick line in FIG. 1). The remaining cells in the group contain probes specific for various mutations leading to the replacement of an amino acid or nucleotide. Such probes form imperfect hybridization duplexes with wild-type DNA.

The list of oligonucleotide sequences immobilized on a biochip is shown in Table 3.

Table 3 List of oligonucleotide sequences immobilized on a biochip SEQ ID No The name of the probe in accordance with Figure 1 Gene / target Probe designation 5'- 3 'sequence 37 507 rpoB Probe for identifying mutations in the rpoB gene, codon 507, wild type TGGCTGGTGCCGAAGA 38 507 rpoB Probe for the identification of mutations in the rpoB gene, codon 507, deletion CTCAGCTGGCTGAAGAA 39 L511 rpoB Probe for the identification of mutations in the rpoB gene, codon 511, wild type TTGGCTCAGCTGGCTG 40 511P rpoB Probe for the identification of mutations in the rpoB gene, codon 511, replacement L> P TTGGCTCGGCTGGCT 41 511R rpoB Probe for the identification of mutations in the rpoB gene, codon 511, replacement L> R TTGGCTCCGCTGGCT 42 S512 rpoB Probe for the identification of mutations in the rpoB gene, codon 512, wild type AATTGGCTCAGCTGGC 43 512T rpoB Probe for the identification of mutations in the rpoB gene, codon 512, replacement S> T AATTGGGTCAGCTGG 44 512R rpoB Probe for the identification of mutations in the rpoB gene, codon 512, replacement S> R AATTGGCGCAGCTG 45 N513 rpoB Probe for the identification of mutations in the rpoB gene, codon 513, wild type TCCATGAATTGGCTCAGCT 46 513L rpoB Probe for the identification of mutations in the rpoB gene, codon 513, replacement N> L CATGAATAGGCTCAGCT 47 513K rpoB Probe for the identification of mutations in the rpoB gene, codon 513, replacement N> K CATGAATTTGCTCAGCT 48 513G rpoB Probe for the identification of mutations in the rpoB gene, codon 513, replacement N> G CATGAATGGGCTCAGCT 49 D516 rpoB Probe for the identification of mutations in the rpoB gene, codon 516, wild type TTGTTCTGGTCCATGAAT fifty 516Y rpoB Probe for the identification of mutations in the rpoB gene, codon 516, replacement D> Y TTGTTCTGGTACATGAAT 51 516G rpoB Probe for the identification of mutations in the rpoB gene, codon 516, replacement D> G TTGTTCTGGCCCATGAAT 52 516V rpoB Probe for the identification of mutations in the rpoB gene, codon 516, replacement D> V TTGTTCTGGACCATGAAT 53 516E rpoB Probe for the identification of mutations in the rpoB gene, codon 516, replacement D> E TTGTTCTGCTCCATGAAT 54 516A rpoB Probe for the identification of mutations in the rpoB gene, codon 516, replacement D> A TTGTTCTGGGCCATGAAT 55 S522 rpoB Probe for the identification of mutations in the rpoB gene, codon 522, wild type TCAACCCCGACAGCG 56 522L rpoB Probe for the identification of mutations in the rpoB gene, codon 522, replacement S> L TCAACCCCAACAGCG 57 H526 rpoB Probe for the identification of mutations in the rpoB gene, codon 526, wild type GGCGCTTGTGGGTCAAC 58 526Y rpoB Probe for the identification of mutations in the rpoB gene, codon 526, replacement H> Y GGCGCTTGTAGGTCAAC 59 526D rpoB Probe for the identification of mutations in the rpoB gene, codon 526, replacement H> D GGCGCTTGTCGGTCAAC 60 526N rpoB Probe for the identification of mutations in the rpoB gene, codon 526, replacement H> N GGCGCTTGTTGGTCAAC 61 526P rpoB Probe for the identification of mutations in the rpoB gene, codon 526, replacement H> P GGCGCTTGGGGGTCTCAC 62 526Q rpoB Probe for the identification of mutations in the rpoB gene, codon 526, replacement H> Q GGCGCTTTTGGGTCAAC 63 526C rpoB Probe for the identification of mutations in the rpoB gene, codon 526, replacement H> C GGCGCTTGCAGGTCAAC 64 526L rpoB Probe for the identification of mutations in the rpoB gene, codon 526, replacement H> L GGCGCTTGAGGGTCAAC 65 526R rpoB Probe for the identification of mutations in the rpoB gene, codon 526, replacement H> R GGCGCTTGCGGGTCTCAC 66 526F rpoB Probe for the identification of mutations in the rpoB gene, codon 526, replacement H> F GGCGCTTGAAGGTCAAC 67 S531 rpoB Probe for the identification of mutations in the rpoB gene, codon 531, wild type CCAGCGCCGACAGTCGG 68 531L rpoB Probe for the identification of mutations in the rpoB gene, codon 531, replacement S> L CCCAGCGCCAACAGTCGG 69 531C rpoB Probe for the identification of mutations in the rpoB gene, codon 531, replacement S> C CCAGCGCACACAGTCGG 70 531W rpoB Probe for the identification of mutations in the rpoB gene, codon 531, replacement S> W CCAGCGCCCACAGTCGG 71 531N rpoB Probe for the identification of mutations in the rpoB gene, codon 531, replacement S> N CCAGCGCCTGCAGTCGG 72 L533 rpoB Probe for identifying mutations in the rpoB gene, codon 533, wild type TGCCCCAGCGCCGACAG 73 L533P rpoB Probe for the identification of mutations in the rpoB gene, codon 533, replacement L> P TGCCCCGGCGCCGACAG 74 S315 katG KatG gene mutation probe, codon 315, wild type CGATCACCAGCGGCAT 75 315T (1) katG Probe for the identification of mutations in the katG gene, codon 315, replacement S> T (option 1) CGATCACCACCGGCAT 76 315T (2) katG Probe for the identification of mutations in the katG gene, codon 315, replacement S> T (option 2) CGATCACCACAGGCATC 77 315N katG Probe for identifying mutations in the katG gene, codon 315, replacement S> N GATCACCAACGGCATC 78 315I katG Probe for the identification of mutations in the katG gene, codon 315, replacement S> I GATCACCATCGGCATC 79 315R (1) katG Probe for the identification of mutations in the katG gene, codon 315, replacement S> R (option 1) GATCACCCGCGGCAT 80 315R (2) katG Probe for identifying mutations in the katG gene, codon 315, replacement S> R (option 2) ATCACCAGAGGCATCG 81 315g katG Probe for the identification of mutations in the katG gene, codon 315, replacement S> G GATCACCGGCGGCAT 82 W328 katG KatG gene mutation probe, codon 328, wild type CGAAATGGGACAACA 83 328g katG KatG gene mutation probe, codon 328, replacement W> G CGAAAGGGGACAACA 84 328L katG KatG gene mutation probe codon 328, replacement W> L CGAAATTGGACAACAG 85 328C katG KatG gene mutation probe, codon 328, replacement W> C CGAAATGCGACAACA 86 I335 katG KatG gene mutation probe, codon 335, wild type TCCTCGAGATCCTGTACG 87 335V katG Probe for the identification of mutations in the katG gene, codon 335, replacement I> V TCCTCGAGGTCCTGTACG 88 t8 inhA Probe for identifying mutations in the inhA gene, position 8, wild type GATAGGTTGTCGGGG 89 8a inhA Probe for identifying mutations in the inhA gene, position 8, replacement T> A GATAGGATGTCGGGG 90 8g inhA Probe for identifying mutations in the inhA gene, position 8, replacement T> G GATAGGGTGTCGGGG 91 c15 inhA Probe for identifying mutations in the inhA gene, position 15, wild type CGGCGAGACGATAGG 92 15t inhA Probe for identifying mutations in the inhA gene, position 15, substitution C> T CGGCGAGATGATAGG 93 16g inhA Probe for identifying mutations in the inhA gene, position 16, substitution C> G GGCGAGGCGATAGG 94 g24 inhA Probe for identifying mutations in the inhA gene, position 24, wild type CCGGCCGCGGCG 95 24t inhA Probe for identifying mutations in the inhA gene, position 24, replacement G> T CCGGCCTCGGCG 96 g6 ahpC Probe for identifying mutations in the ahpC gene, position 6, wild type GCACGATGGAATGTCGC 97 6a ahpC Probe to identify mutations in the ahpC gene, position 6, replacement G> A GCACGATAGAATGTCGCA 98 c10 ahpC Probe to identify mutations in the ahpC gene, position 10, wild type TCACGGCACGATGGAA 99 10t ahpC Probe for identifying mutations in the ahpC gene, position 10, substitution C> T TCACGGCATGATGGAA one hundred 10a ahpC Probe for identifying mutations in the ahpC gene, position 10, substitution C> A TCACGGCAAGATGGAA 101 g9 ahpC Probe for identifying mutations in the ahpC gene, position 9, wild type CACGGCACGATGGAAT 102 9a ahpC Probe for identifying mutations in the ahpC gene, position 9, replacement G> A CACGGCACAATGGAAT 103 c12 ahpC Probe to identify mutations in the ahpC gene, position 12, wild type CTTCACGGCACGATGG 104 12t ahpC Probe to identify mutations in the ahpC gene, position 12, substitution C> T ACTTCACGGTACGATGGA 105 IS IS6110 Mycobacterium tuberculosis complex DNA identification probe CCGGAGCTGCGTGAGCG 106 N296 embB EmbB gene mutation probe, codon 296, wild type GGCGCGAATTCGTCG 107 296H embB Probe for the identification of mutations in the embB gene, codon 296, replacement N> H GCGCGCATTCGTCG 108 S297 embB EmbB gene mutation probe, codon 297, wild type CGCGAATTCGTCGGACG 109 297A embB Probe for the identification of mutations in the embB gene, codon 297, replacement S> A CGCGAATGCGTCGGAC 110 M306 embB EmbB gene mutation probe, codon 306, wild type CCTGGGCATGGCCCGAG 111 306V embB Probe for the identification of mutations in the embB gene, codon 306, replacement M> V CCTGGGCGTGGCCCGAG 112 306L embB Probe for the identification of mutations in the embB gene, codon 306, replacement M> L CCTGGGCCTGGCCCGAG 113 306I1 embB Probe for the identification of mutations in the embB gene, codon 306, replacement M> I (option 1) CCTGGGCATAGCCCGAG 114 306I2 embB Probe for the identification of mutations in the embB gene, codon 306, replacement M> I (option 2) CCTGGGCATCGCCCGAG 115 306I3 embB Probe for the identification of mutations in the embB gene, codon 306, replacement M> I (option 3) CCTGGGCATTGCCCGAG 116 V309 embB EmbB gene mutation probe, codon 309, wild type GCCCGAGTCGCCG 117 309F embB Probe for the identification of mutations in the embB gene, codon 309, replacement V> S GCCCGATTCGCCGA 118 A313 embB EmbB gene mutation probe, codon 313, wild type GACCACGCCGGCTAC 119 313V embB Probe for the identification of mutations in the embB gene, codon 313, replacement A> V CGACCACGTCGGCTACA 120 Y319 embB EmbB gene mutation probe, codon 319, wild type ATGTCCAACTATTTCCGCTGG 121 319C embB Probe for the identification of mutations in the embB gene, codon 319, substitution Y> C ATGTCCAACTGTTTCCGCTG 122 319S embB Probe for the identification of mutations in the embB gene, codon 319, replacement Y> S ATGTCCAACTCTTTCCGCTG 123 319D embB Probe for the identification of mutations in the embB gene, codon 319, replacement Y> D ATGTCCAACGATTTCCGCTG 124 D328 embB EmbB gene mutation probe, codon 328, wild type CCCGGAGGATCCCTTCG 125 328Y embB Probe for the identification of mutations in the embB gene, codon 328, replacement D> Y CCCGGAGTATCCCTTCGG 126 328g embB Probe for the identification of mutations in the embB gene, codon 328, replacement D> G CCGGAGGGTCCCTTCG 127 D354 embB EmbB gene mutation probe, codon 354, wild type CCTACCAGACCTGGCCGCCGG 128 354A embB Probe for the identification of mutations in the embB gene, codon 354, replacement D> A CCTACCAGCCCTGGCCGCGG 129 E378 embB EmbB gene mutation probe, codon 378, wild type GCGGTGGAGGCCAGCA 130 378A embB Probe for the identification of mutations in the embB gene, codon 378, replacement E> A CGGTGGCGGCCAGC 131 G406 embB EmbB gene mutation probe, codon 406, wild type CCGGAGGGCATCATC 132 406A embB Probe for the identification of mutations in the embB gene, codon 406, replacement G> A CCGGAGGCCATCATC 133 406D embB Probe for the identification of mutations in the embB gene, codon 406, replacement G> D CCGGAGGACATCATCG 134 406C embB Probe for the identification of mutations in the embB gene, codon 406, replacement G> C CCGGAGTGCATCATCG 135 406S embB Probe for the identification of mutations in the embB gene, codon 406, replacement G> S CCGGAGAGCATCATCG 136 Q497 embB EmbB gene mutation probe, codon 457, wild type CGCCGACCAGACCCT 137 497R embB Probe for the identification of mutations in the embB gene, codon 457, substitution Q> R CGCCGACCGGACCCT 138 497K embB Probe for the identification of mutations in the embB gene, codon 457, substitution Q> K CGCCGACAAGACCCT 139 497P embB Probe for the identification of mutations in the embB gene, codon 457, substitution Q> P CGCCGACCCGACCCT 140 H70 gyrA GyrA gene mutation probe, codon 70, wild type GACCGCAGCCACGCCAAGTCG 141 70R gyrA GyrA gene mutation probe, codon 70, replacement H> R ACCGCAGCCGCGCCAAGTC 142 A74 gyrA GyrA gene mutation probe, codon 74, wild type CCAAGTCGGCCCGGTCGG 143 74S gyrA GyrA gene mutation probe, codon 74, replacement A> S GCCAAGTCGTCCCGGTCGG 144 T80 gyrA GyrA gene mutation probe, codon 80, wild type TGCCGAGACCATGGGC 145 80A gyrA GyrA gene mutation probe, codon 80, replacement T> A GCCGAGGCCATGGGC 146 G88 gyrA GyrA gene mutation probe, codon 88, wild type CCCGCACGGCGACGCG 147 88A gyrA GyRA gene mutation probe, codon 88, replacement G> A CCCGCACGCCGACGCG 148 88C gyrA GyRA gene mutation probe, codon 88, replacement G> C CCCGCACTGCGACGCG 149 A90 gyrA GyrA gene mutation probe, codons 90, 91, wild type CGACGCGTCGATCTACGA 150 90V gyrA GyrA gene mutation probe, codons 90, 91, A> V substitution, wild type CGACGTGTCGATCTACGA 151 90g gyrA GyrA gene mutation probe, codons 90, 91, A> G substitution, wild type CGACGGGTCGATCTACGA 152 90 * gyrA GyrA gene mutation probe, codons 90, 91, wild type, S> P CGACGCGCCGATCTACG 153 90V * gyrA Probe for identifying mutations in the gyrA gene, codons 90, 91, substitutions A> V, S> P CGACGTGCCGATCTACGA 154 90G * gyrA Probe for identifying mutations in the gyrA gene, codons 90, 91, substitutions A> G, S> P CGACGGGCCGATCTACG 155 D94 gyrA GyrA gene mutation probe, codons 94, 95, wild type CGATCTACGACAGCCTG 156 94H gyrA GyrA gene mutation probe, codons 94, 95, replacement D> H CGATCTACCACAGCCTG 157 94A gyrA GyrA gene mutation probe, codons 94, 95, substitution D> A, wild type GATCTACGCCAGCCTG 158 94N gyrA GyrA gene mutation probe, codons 94, 95, substitution D> N, wild type TCGATCTACAACAGCCTG 159 94g gyrA GyrA gene mutation probe, codons 94, 95, substitution D> G, wild type GATCTACGGCAGCCTG 160 94Y gyrA GyrA gene mutation probe, codons 94, 95, substitution D> Y, wild type TCGATCTACTACAGCCTG 161 94V gyrA GyrA gene mutation probe, codons 94, 95, substitution D> V, wild type CGATCTACGTCAGCCTG 162 D94 * gyrA GyrA gene mutation probe, codons 94, 95, wild type, replacement S> T TCTACGACACCCTGGT 163 94H * gyrA GyrA gene mutation probe, codons 94, 95, replacement D> H, replacement S> T CGATCTACCACACCCTG 164 94A * gyrA GyrA gene mutation probe, codons 94, 95, replacement D> A, replacement S> T TCTACGCCACCCTGGT 165 94N * gyrA GyrA gene mutation probe, codons 94, 95, replacement D> N, replacement S> T TCGATCTACAACACCCTG 166 94G * gyrA GyrA gene mutation probe, codons 94, 95, replacement D> G, replacement S> T GATCTACGGCACCCTG 167 94Y * gyrA GyrA gene mutation probe, codons 94, 95, replacement D> Y, replacement S> T CGATCTACTACACCCTG 168 94V * gyrA GyrA gene mutation probe, codons 94, 95, replacement D> V, replacement S> T CGATCTACGTCACCCTG 169 P102 gyrA GyrA gene mutation probe, codon 102, wild type GCCCAGCCCTGGTCGCT 170 102H gyrA GyrA gene mutation probe codon 102, substitution P> H GGCCCAGCACTGGTCGC 171 R485 gyrB GyrB gene mutation probe, codon 485, wild type GCCGATTGCCGTTCCACG 172 485C gyrB GyrB gene mutation probe, codon 485, replacement R> C GCCGATTGCTGTTCCACG 173 485H gyrB GyrB gene mutation probe, codon 485, replacement R> H CCGATTGCCATTCCACG 174 485L gyrB GyrB gene mutation probe, codon 485, replacement R> L GCCGATTGCCTTTCCACG 175 S486 gyrB GyrB gene mutation probe, codon 486, wild type CGATTGCAGTTCCACGGATCCGC 176 486F gyrB GyrB gene mutation probe, codon 486, replacement S> F CGATTGCAGTTTCACGGATCCGCG 177 D500 gyrB GyrB gene mutation probe, codon 500, wild type GTAGAAGGTGACTCGGCCG 178 500H gyrB GyrB gene mutation probe, codon 500, replacement D> H GTAGAAGGTCACTCGGCCG 179 500N gyrB GyrB gene mutation probe, codon 500, replacement D> N CGTAGAAGGTAACTCGGCCG 180 500A gyrB GyrB gene mutation probe, codon 500, replacement D> A TAGAAGGTGCCTCGGCCG 181 G509 gyrB GyrB gene mutation probe, codon 509, wild type GCAAAAAGCGGTCGCGATTC 182 509C gyrB GyrB gene mutation probe, codon 509, replacement G> C TGCAAAAAGCTGTCGCGATTC 183 509A gyrB GyrB gene mutation probe, codon 509, replacement G> A GCAAAAAGCGCTCGCGATTC 184 I525 gyrB GyrB gene mutation probe, codon 525, wild type CGGCAAGATCATCAATGTGGA 185 525L gyrB GyrB gene mutation probe, codon 525, replacement I> L CGGCAAGATCCTCAATGTGG 186 D533 gyrB GyrB gene mutation probe, codon 533, wild type GCGCATCGACCGGGTG 187 533A gyrB GyrB gene mutation probe, codon 533, replacement D> A CGCATCGCCCGGGTG 188 N538 gyrB GyrB gene mutation probe, codon 538, wild type GTGCTAAAGAACACCGAA 189 538D gyrB GyrB gene mutation probe, codon 538, replacement N> D TGCTAAAGGACACCGAA 190 538Y gyrB GyrB gene mutation probe, codon 538, replacement N> Y GTGCTAAAGTACACCGAAG 191 538K gyrB GyrB gene mutation probe, codon 538, replacement N> K GTGCTAAAGAAAACCGAA 192 538T gyrB GyrB gene mutation probe, codon 538, replacement N> T TGCTAAAGACCACCGAA 193 T539 gyrB GyrB gene mutation probe, codon 539, wild type CTAAAGAACACCGAAGTTC 194 539I gyrB GyrB gene mutation probe, codon 539, replacement T> I CTAAAGAACATCGAAGTTCA 195 539N gyrB GyrB gene mutation probe, codon 539, replacement T> N GCTAAAGAACAACGAAGTT 196 539P gyrB GyrB gene mutation probe, codon 539, replacement T> P CTAAAGAACCCCGAAGTT 197 E540 gyrB GyrB gene mutation probe, codon 540, wild type AACACCGAAGTTCAGGC 198 540D (1) gyrB The probe for the identification of mutations in the gyrB gene, codon 540, replacement E> D (option 1) AACACCGATGTTCAGGC 199 540D (2) gyrB Probe for identifying mutations in the gyrB gene, codon 540, replacement E> D (option 2) AACACCGACGTTCAGG 200 540V gyrB GyrB gene mutation probe, codon 540, replacement E> V AACACCGTAGTTCAGGC 201 A543 gyrB GyrB gene mutation probe, codon 543, wild type AGTTCAGGCGATCATCA 202 543T gyrB GyrB gene mutation probe, codon 543, replacement A> T AAGTTCAGACGATCATCA 203 543V gyrB GyrB gene mutation probe, codon 543, replacement A> T AGTTCAGGTGATCATCA 204 1401 rrs Probe for identifying mutations in the rrs gene, position 1401, wild type CGCCCGTCACGTCATGAA 205 1401g rrs Probe for identifying mutations in the rrs gene, position 1401, replacement C> G GCCCGTCGCGTCATGAA 206 1402t rrs Probe for the identification of mutations in the rrs gene, position 1401, replacement C> T CGCCCGTCATGTCATGAAA 207 1402a rrs Probe for identifying mutations in the rrs gene, position 1401, substitution C> A CGCCCGTCAAGTCATGAAA 208 1484 rrs Probe for identifying mutations in the rrs gene, position 1484, wild type GATTGGGACGAAGTCGTAACA 209 1484t rrs Probe for identifying mutations in the rrs gene, position 1484, replacement G> T GATTGGGACTAAGTCGTAACA 210 -10 rrs Probe for identifying mutations in the eis gene, position 10, wild type GCATATGCCACAGTCGGATT 211 s-14t eis Probe for the identification of mutations in the eis gene, position 14, substitution C> T GGCATATGCTACAGTCGGATT 212 a-13g eis Probe for the identification of mutations in the eis gene, position 13, replacement A> G GCATATGCCGCAGTCGGATT 213 c-12t eis Probe for the identification of mutations in the eis gene, position 12, substitution C> T GCATATGCCATAGTCGGATTC 214 g-10a eis Probe for the identification of mutations in the eis gene, position 10, substitution G> A CATATGCCACAATCGGATTCTG 215 g-10c eis Probe for the identification of mutations in the eis gene, position 10, substitution G> C ATATGCCACACTCGGATTCT 216 -35 eis Probe for the identification of mutations in the eis gene, position 35, wild type CGTAATATTCACGTGCACGTAGC 217 g-37t eis Probe for the identification of mutations in the eis gene, position 37, replacement G> T TAATATTCACTTGCACGTAGCC 218 309 Rv0129c LAM genotype probe CGGCCTTCGAGGAGTACTACC 219 309a Rv0129c LAM genotype probe CGGCCTTCGAAGAGTACTACCA 220 321 Rv0557 Probe for determination of belonging to the Euro-American line TGGCTACGGTGGACTCCAT 221 321c Rv0557 Probe for determination of belonging to the Euro-American line GGCTACGGCGGACTCC 222 455 Rv0557 Haarlem genotype probe CACTTGCATCGCCTGGCT 223 455c Rv0557 Haarlem genotype probe CACTTGCATCCCCTGGCT 224 12 Rv1811 Ural genotype probe GCAAACGCTGACTGTCGC 225 g12a Rv1811 Ural genotype probe GCAAACGCTAACTGTCGCC 226 191 Rv2629 Beijing Genotype Identification Probe GGTGCGGGATTCTCGACC 227 191c Rv2629 Beijing Genotype Identification Probe TGCGGGCTTCTCGACC 228 g757 oxcA / Rv0118c Genotype identification probe Beijing B0 ATGTCGATGGCCAAGGGG 229 757t oxcA / Rv0118c Genotype identification probe Beijing B0 GATGTCGATGTCCAAGGGG

Example 3. Analysis of a laboratory strain of M. tuberculosis H37Rv sensitive to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin / capreomycin, ethambutol.

Processing the culture of the strain M. tuberculosis H37Rv, DNA extraction was performed, amplification of the studied fragments of the mycobacterial genome and hybridization on the biochip were performed as described in Example 1.

The hybridization results are shown in FIG. 2.

According to the algorithm for processing the results of hybridization, the signal value in the IS cell exceeded the average background signal by more than 4 times. Therefore, the presence of mycobacterium tuberculosis complex DNA was found in this sample.

An analysis of a group of cells with probes specific for Rv0557 polymorphism revealed the presence of the wild type, which means that the strain belongs to the Euro-American line. Analysis of cells with probes specific for Rv0557_455, Rv0129c, Rv1811 polymorphisms showed the presence of the wild type. Thus, it was not possible to establish the genotype for this strain, which is true, since the H37Rv strain is laboratory and forms a separate phylogenetic branch.

An analysis of groups of cells with probes specific for resistance determinants showed that in each group a perfect hybridization duplex was registered in a cell corresponding to the wild type. Thus, no mutations were detected in any of the studied fragments of the M. tuberculosis H37Rv genome, which means the sensitivity of this strain to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin, capreomycin, ethambutol. This result coincides with the microbiological data on the drug sensitivity of the M. tuberculosis H37Rv strain.

Example 4. Analysis of a clinical sample containing a strain of M. tuberculosis of the Ural genotype, which is resistant to isoniazid.

A clinical specimen (sputum) obtained from a patient with microscopic evidence of acid-resistant bacteria (BK +) was divided into 2 parts, one of which, after decontamination (N-acetyl-L-cysteine and NaOH) and neutralization, was transferred for microbiological studies . Mycobacterium tuberculosis complex was identified by biochemical tests (Kent PT, Kubica GP. Public health mycobacteriology. A guide for level III laboratory. Atlanta, GA: Centers for Disease Control and Prevention, 1985). Tests for resistance to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin, capreomycin, ethambutiol were carried out using the Bactec MGIT 960 automated system (Becton Dickinson, USA). The second part of the sputum sample was analyzed according to the invention, as described in Examples 1 and 2.

In FIG. 3 shows the result of a DNA analysis isolated from a given clinical sample and investigated according to the invention.

According to the algorithm for processing the results of hybridization, the signal value in the IS cell exceeded the average background signal by more than 4 times. Therefore, the presence of mycobacterium tuberculosis complex DNA was found in this sample.

An analysis of a group of cells with probes specific for Rv0557 polymorphism revealed the presence of the wild type, which means that the strain belongs to the Euro-American line. An analysis of a group of cells with probes specific for Rv1811 polymorphism (circled by a dashed line) showed the presence of a substitution Rv1811_12G> A. Thus, the genotype of this strain was identified as Ural.

An analysis of groups of cells with probes specific for resistance determinants showed that in each group of cells corresponding to variable amino acid 315 in the katG gene, a perfect hybridization duplex was registered in the cell corresponding to the mutation Ser 315> Thr (option 1). Therefore, the DNA of this strain contains such a mutation and the strain is resistant to isoniazid. In other groups of cells, perfect hybridization duplexes were registered only in cells corresponding to the wild type. Thus, no mutations were detected in any of the studied fragments of the rpoB, gyrA, gyrB, eis, rrs, embB genes of this strain, which means the sensitivity of this strain to rifampicin, fluoroquinolones, kanamycin, amikacin, capreomycin, ethambutol.

Microbiological and biochemical tests confirmed the presence of M. tuberculosis in sputum. Resistance to isoniazid was confirmed by culture growth on the corresponding specific medium with isoniazid, in the absence of growth on media with other drugs. Thus, the culture method recorded the presence in the clinical sample of a strain of M. tuberculosis that is isoniazide monoresistant, which completely coincides with the results obtained by the method of the present invention.

Example 5. Analysis of a clinical sample containing a strain of M. tuberculosis genotype Beijing B0 / W148, which is resistant to rifampicin, isoniazid, ethambutol.

A clinical sample (bronchoalveolar lavage) obtained from a patient with microscopic evidence of acid-resistant bacteria (BK +) was divided into 2 parts, one of which, after decontamination (N-acetyl-L-cysteine and NaOH) and neutralization, was transferred for microbiological research. Mycobacterium tuberculosis complex was identified by biochemical tests (Kent PT, Kubica GP. Public health mycobacteriology. A guide for level III laboratory. Atlanta, GA: Centers for Disease Control and Prevention, 1985). Tests for resistance to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin, capreomycin, ethambutiol were carried out using the Bactec MGIT 960 automated system (Becton Dickinson, USA). The second part of the sputum sample was analyzed according to the invention, as described in Examples 1 and 2.

In FIG. 4 shows the result of analysis of DNA isolated from a given clinical sample and investigated according to the invention.

According to the algorithm for processing the results of hybridization, the signal value in the IS cell exceeded the average background signal by more than 4 times. Therefore, the presence of mycobacterium tuberculosis complex DNA was found in this sample.

An analysis of a group of cells with probes specific for Rv0557 polymorphism (circled by a dashed line) showed the presence of Rv0557_321T> C polymorphism, which means that the strain does not belong to the Euro-American line. An analysis of a group of cells with probes specific for Rv2629 polymorphism (circled by a dashed line) showed that this strain belongs to the Beijing family. The established polymorphism Rv0118c_G> T in the oxcA gene (cells are surrounded by a dashed line) made it possible to attribute this strain to the Beijing B0 / W148 genotype.

An analysis of cell groups with probes specific for resistance determinants revealed the mutation Ser531> Leu in the rpoB gene (arrow 1 in Fig. 4), leading to rifampicin resistance; mutation Ser315> Thr (option 2) in the katG gene (arrow 2), leading to isoniazid resistance; mutation Met 306> Ile (option 1) in the embB gene (arrow 3), associated with resistance to ethambutol. In other groups of cells, perfect hybridization duplexes were registered only in cells corresponding to the wild type. Thus, no mutations were detected in any of the studied fragments of the genes gyrA, gyrB, eis, rrs of this strain, which means the sensitivity of this strain to fluoroquinolones and kanamycin, amikacin / capreomycin.

Microbiological and biochemical tests confirmed the presence of M. tuberculosis in a clinical sample. Resistance to rifampicin, isoniazid, ethambutol was confirmed by culture growth on specific media with the appropriate chemotherapy, in the absence of growth on media with fluoroquinolones, kanamycin, amikacinui and capreomycin. Thus, the culture method recorded the presence in the clinical sample of a strain of M. tuberculosis that has multidrug resistance (to rifampicin and isoniazid) and is additionally resistant to ethambutol, which completely coincides with the results obtained by the method of the present invention.

Example 6. Analysis of a clinical sample containing a strain of M. tuberculosis of the Beijing genotype, which is resistant to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin / capreomycin and ethambutol.

The clinical sample (blood) obtained from the patient, with the presence of acid-resistant bacteria (BK +) confirmed by microscopy, was divided into 2 parts, one of which, after decontamination (N-acetyl-L-cysteine and NaOH) and neutralization, was transferred for microbiological studies . Mycobacterium tuberculosis complex was identified by biochemical tests (Kent PT, Kubica GP. Public health mycobacteriology. A guide for level III laboratory. Atlanta, GA: Centers for Disease Control and Prevention, 1985). Tests for resistance to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin, capreomycin, ethambutiol were carried out using the Bactec MGIT 960 automated system (Becton Dickinson, USA). The second part of the sputum sample was analyzed according to the invention, as described in Examples 1 and 2.

In FIG. 5 shows the result of a DNA analysis isolated from a given clinical sample and investigated according to the invention.

According to the algorithm for processing the results of hybridization, the signal value in the IS cell exceeded the average background signal by more than 4 times. Therefore, the presence of mycobacterium tuberculosis complex DNA was found in this sample.

An analysis of a group of cells with probes specific for Rv0557 polymorphism (circled by a dashed line) showed the presence of Rv0557_321T> C polymorphism, which means that the strain does not belong to the Euro-American line. An analysis of a group of cells with probes specific for Rv2629 polymorphism (circled by a dashed line) showed that this strain belongs to the Beijing family.

The analysis of cell groups with probes specific for resistance determinants revealed the mutation Ser531> Leu in the rpoB gene (arrow 1 in Figure 5), leading to rifampicin resistance; mutation Asp94> Ala in the gyrA gene (arrow 2), leading to resistance to fluoroquinolones; mutation Ser315> Arg (option 1) in the katG gene (arrow 3), leading to isoniazid resistance; nucleotide substitution g37> t in the regulatory region of the eis gene (arrow 4), associated with resistance to kanamycin, amikacin; mutation Asp 354> Ala in the embB gene (arrow 5), associated with resistance to ethambutol. Thus, the determinants of resistance for each of the analyzed drugs were found in the DNA of this strain.

Microbiological and biochemical tests confirmed the presence of M. tuberculosis in the blood. Resistance to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin, capreomycin, ethambutol was confirmed by cultural growth on specific media with the appropriate chemotherapeutic agents. Thus, the culture method recorded the presence in the clinical sample of a strain of M. tuberculosis with extensive drug resistance, which completely coincides with the results obtained by the method of the present invention.

Example 7. Analysis of a clinical sample containing a strain of M. tuberculosis genotype LAM, which is resistant to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin / capreomycin and ethambutol.

A clinical specimen (sputum) obtained from a patient with microscopic evidence of acid-resistant bacteria (BK +) was divided into 2 parts, one of which, after decontamination (N-acetyl-L-cysteine and NaOH) and neutralization, was transferred for microbiological studies . Mycobacterium tuberculosis complex was identified by biochemical tests (Kent PT, Kubica GP. Public health mycobacteriology. A guide for level III laboratory. Atlanta, GA: Centers for Disease Control and Prevention, 1985). Tests for resistance to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin, capreomycin, ethambutiol were carried out using the Bactec MGIT 960 automated system (Becton Dickinson, USA). The second part of the sputum sample was analyzed according to the invention, as described in Examples 1 and 2.

In FIG. 6 shows the result of analysis of DNA isolated from a given clinical sample and investigated according to the invention.

According to the algorithm for processing the results of hybridization, the signal value in the IS cell exceeded the average background signal by more than 4 times. Therefore, the presence of mycobacterium tuberculosis complex DNA was found in this sample.

An analysis of the group of cells, with probes specific for Rv0557 polymorphism, showed the presence of the wild-type Rv0557_321 polymorphism, which meant the strain belonged to the Euro-American line. An analysis of a group of cells with probes specific for Rv0129c polymorphism (circled by a dashed line) showed that this strain belongs to the LAM family.

The analysis of cell groups with probes specific for resistance determinants revealed the Asp515> Val mutation in the rpoB gene (arrow 1 in Fig. 6), leading to rifampicin resistance; mutation Ala90> Val in the gyrA gene (arrow 2), leading to resistance to fluoroquinolones; c15> t nucleotide substitution in the regulatory region of the inhA gene (arrow 3) and the Ser315> Thr mutation (option 1) in the katG gene (arrow 4), leading to isoniazid resistance; mutation a1401> g in the rrs gene (arrow 5), leading to resistance to kanamycin, amikacin and capreomycin; mutations Met306> Ile (option 1) and Gly406> Asp (arrows 6 and 7, respectively) in the embB gene, associated with ethambutol resistance. Thus, the determinants of resistance for each of the analyzed drugs were found in the DNA of this strain.

Microbiological and biochemical tests confirmed the presence of M. tuberculosis in sputum. Resistance to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin, capreomycin, ethambutol was confirmed by cultural growth on specific media with the appropriate chemotherapeutic agents. Thus, the culture method recorded the presence in the clinical sample of a strain of M. tuberculosis with extensive drug resistance, which completely coincides with the results obtained by the method of the present invention.

Example 8. Analysis of a clinical sample not containing mycobacterium tuberculosis complex.

A clinical sample (exudate) obtained from a patient with microscopic evidence of acid-resistant bacteria (BK +) was divided into 2 parts, one of which, after decontamination (N-acetyl-L-cysteine and NaOH) and neutralization, was transferred for microbiological studies . Mycobacterium tuberculosis complex was identified by biochemical tests (Kent PT, Kubica GP. Public health mycobacteriology. A guide for level III laboratory. Atlanta, GA: Centers for Disease Control and Prevention, 1985). Tests for resistance to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin, capreomycin, ethambutiol were carried out using the Bactec MGIT 960 automated system (Becton Dickinson, USA). The second part of the sputum sample was analyzed according to the invention, as described in Examples 1 and 2.

In FIG. 7 shows the result of a DNA analysis isolated from a given clinical sample and investigated according to the invention.

According to the algorithm for processing the results of hybridization, the signal value in the IS cell was close to the background value, i.e. exceeded the average signal in empty gel cells by no more than 20%. Thus, it was found that the test sample does not contain the DNA of the Mycobacterium tuberculosis complex, and, therefore, analysis to establish the genotype and determinants of resistance was not carried out.

Microbiological and biochemical tests confirmed the presence in the clinical sample of M.avium, not belonging to the mycobacterium tuberculosis complex. Thus, the results obtained by the method of the present invention and the reference method completely coincided.

Thus, the presented invention allows to quickly identify the DNA of the causative agent of tuberculosis - mycobacterium tuberculosis complex in clinical samples, to establish the genotype of the pathogen and at the same time to identify the genetic determinants of resistance to the most effective drugs of the first and second row - rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin, capreomycin ethambutol. The method of the present invention compares favorably with existing analogues with the largest number of simultaneously determined determinants of resistance, making it possible to establish anti-TB drugs effective for each particular patient. Unlike test systems based on hydrogel biochips of the previous generation, the proposed option of a single-stage multiplex PCR significantly reduces the likelihood of cross-contamination and reduces the time and labor involved in the analysis. The identification of clinically significant genotypes, especially the Beijing family, associated with increased virulence and transmissibility, will allow measures to be taken to prevent the spread of such strains. The present invention also allows for epidemiological genotyping, establishing the genotypes of the most common families in the Russian Federation.

Although the preferred embodiments of the present invention and their advantages have been described in detail above, a person skilled in the art will be able to make various changes, additions, or, conversely, omit something, without going beyond the scope of this invention, which are defined by the appended claims.

Claims (9)

1. A method for detecting tuberculosis pathogen DNA with the simultaneous establishment of its genotype and determination of the genetic determinants of resistance to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin, capreomycin, ethambutol, including:
(a) - multiplex amplification of rpoB, katG, inhA, ahpC, gyrA, gyrB, rrs, eis, embB genes, mobile element IS6110, fragments of the mycobacterial genome containing genotype-specific single nucleotide polymorphisms Rv0557, Rv0129c, Rv1811, Rv1829, Rv1829, Rv2629, simultaneous fluorescence labeling using the genomic DNA of mycobacterium tuberculosis complex as a template and a set of primers whose sequences are represented by SEQ ID NO: 1-36, to obtain fluorescently labeled fragments of the mycobacterial genome;
(b) - providing an oligonucleotide microchip for identifying the DNA of mycobacterium tuberculosis complex with the simultaneous establishment of the Beijing, Beijing BO, LAM, Haarlem, Ural genotypes and the determination of mutations responsible for the resistance of the tuberculosis pathogen to rifampicin, isoniazid, fluoroquinolones, kanamycin etamolacin, , which is a substrate containing many discrete elements, in each of which a unique oligonucleotide probe is immobilized, having a sequence complementary to a single chain fragment obtained in step (a) and selected from the group consisting of: a) corresponding sequences of a fragment of the mobile element IS6110; b) the corresponding sequence of the fragment, including the Rv0557 polymorphism characteristic of the Euro-American line; c) the corresponding sequence of the fragment, including the Rv0557 polymorphism characteristic of the Haarlem genotype, with established affiliation with the Euro-American line; d) the corresponding sequence of the fragment, including the Rv0129c polymorphism characteristic of the LAM genotype, with established affiliation with the Euro-American line; e) the corresponding sequence of the fragment, including the Rv1811 polymorphism characteristic of the Ural genotype, with established affiliation with the Euro-American line; f) the corresponding sequence of the fragment, including the Rv2629 polymorphism characteristic of the Beijing family, in the absence of belonging to the Euro-American line; g) the corresponding sequence of the fragment including the Rv0118c polymorphism in the oxA gene, characteristic of the Beijing B0 / W148 genotype with the established Beijing family; h) the corresponding sequences of the wild-type rpoB gene fragment; i) the corresponding sequence of the fragment of the mutant variant of the rpoB gene, leading to the resistance of microorganisms to rifampicin; i) the corresponding sequences of the wild-type katG gene fragment; k) the corresponding sequence of the fragment of the mutant variant of the katG gene, leading to the resistance of microorganisms to isoniazid; k) the corresponding sequences of the wild-type inhA gene fragment; m) the corresponding sequence of the fragment of the mutant variant of the inhA gene, leading to the resistance of microorganisms to isoniazid; m) the corresponding sequences of the wild-type ahpC gene fragment; o) the corresponding sequence of the fragment of the mutant variant of the ahpC gene, leading to the resistance of microorganisms to isoniazid; o) the corresponding sequences of the wild-type gyrA gene fragment; p) the corresponding sequence of the fragment of the mutant variant of the gyrA gene, leading to the resistance of microorganisms to fluoroquinolones; c) the corresponding sequences of the wild-type gyrB gene fragment; r) the corresponding sequence of the fragment of the mutant variant of the gyrB gene, leading to the resistance of microorganisms to fluoroquinolones; s) the corresponding sequence of the fragment of the wild-type eis gene; f) the corresponding sequence of the fragment of the mutant variant of the eis gene, leading to the resistance of microorganisms to kanamycin, amikacin and capreomycin; x) the corresponding sequences of the wild-type rrs gene fragment; c) the corresponding sequence of the fragment of the mutant variant of the rrs gene, leading to the resistance of microorganisms to kanamycin, amikacin and capreomycin; h) the corresponding sequences of the wild-type embB gene fragment; sh) the corresponding sequence of the fragment of the mutant variant of the embB gene, leading to the resistance of microorganisms to ethambutol; wherein the sequences of immobilized oligonucleotide probes are represented by SEQ ID NO: 37-229;
(c) - hybridization of the amplified fluorescently-labeled products obtained in stage (a) on an oligonucleotide microchip with the formation of duplexes with immobilized probes under conditions providing a resolution of one nucleotide between perfect and imperfect duplexes formed as a result of hybridization;
(d) - recording the results of hybridization on an oligonucleotide microchip, carried out in stage (c) using a portable fluorescence analyzer and software, which allows the use of software processing of signal intensities with subsequent interpretation of the results, which is carried out in two stages: at the first stage, the signals are analyzed a microchip cell containing an oligonucleotide probe specific for a fragment of the mobile element IS6110, characteristic of the mycobacterium tuberculosis complex, with the aim of aruzheniya DNA of Mycobacterium tuberculosis; in the presence of a signal in this cell, i.e. in the case of detection of mycobacterial DNA, in the second stage, microarray elements containing genotype-specific oligonucleotide probes that study the polymorphisms Rv0557, Rv0129c, Rvl811, Rv2629, Rv0118c, and elements in which oligonucleotide probes are detected in inhA, ahpC, gyrA, gyrB, rrs, eis, embB, thereby establishing the genotype of the causative agent of tuberculosis and determining the genetic determinants of resistance to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin, capreomycin, ethambutol. 2. The method according to claim 1, in which the amplification of fragments of the mycobacterial genome is carried out using DNA isolated from the material of the clinical sample (sputum, exudate, bronchoalveolar lavage, blood) or a pre-grown culture of microorganisms. 3. The method according to p. 1, characterized in that at the stage (a) for fluorescent labeling of the obtained PCR fragments using fluorescently-labeled deoxyuridine triphosphate. 4. The method according to p. 1, characterized in that at stage (d) in the second stage of interpreting the results when analyzing microarray elements containing genotype-specific oligonucleotide probes, if the wild-type is established by the Rv0557_321 polymorphism, the strain is assigned to the Euro-American line, with further identification of the Haarlem genotype (by Rv0557_455 polymorphism), or the LAM genotype (by Rv0129c polymorphism), or Ural (by Rv1811 polymorphism); when another variant of Rv0557_321 polymorphism is detected, the strain is assigned to a different, non-Euro-American line, with further identification of the Beijing family (by Rv2629 polymorphism) and determination of the B0 / W148 genotype for the Beijing family (by Rv0118 polymorphism). 5. The method according to p. 1, characterized in that in stage (d) in the second stage of interpreting the results, groups of elements containing oligonucleotide probes specific to one of the rpob, katG, inhA, ahpC, gyrA, gyrB, rrs, eis genes are analyzed , embB; each group includes an element containing a wild-type probe, and elements containing probes corresponding to mutant gene variants. If the maximum signal in the group is registered in an element containing a wild-type probe, then it is believed that the DNA sample of the tuberculosis causative agent does not have mutations by the given amino acid / nucleotide position of this gene. If the maximum signal in the group is registered in the element containing the probe, the sequence of which is specific for the mutant variant of one of the genes, then it is believed that for a given amino acid / nucleotide position of this gene, the studied DNA sample of the tuberculosis pathogen has an amino acid / nucleotide substitution, which is a genetic determinant of resistance. 6. The method according to claim 1, in which the interpreted results can be applied to confirm the clinical diagnosis of tuberculosis, personalize the choice of effective chemotherapy drugs of the first and second row, establish clinically significant genotypes and the goals of epidemiological genotyping. 7. Oligonucleotide microchip used in the method of detecting tuberculosis pathogen DNA with simultaneous determination of its genotype and determination of genetic determinants of resistance to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin, capreomycin, ethambutol, characterized in that it is a hydrogel substrate chemically or photoinduced copolymerization process and containing immobilized oligonucleotide probes, the sequences of which are represented by SEQ ID NO: 37-229. 8. A set of specific primers for the implementation of a method for detecting tuberculosis pathogen DNA with the simultaneous establishment of its genotype and determination of the genetic determinants of resistance to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin, capreomycin, ethambutol, and primer sequences are presented in SEQ ID NO: 1-36. 9. A set of oligonucleotide probes used to obtain an oligonucleotide microchip used in a method for detecting tuberculosis pathogen DNA with simultaneous determination of its genotype and determination of genetic determinants of resistance to rifampicin, isoniazid, fluoroquinolones, kanamycin, amikacin, capreomycin nucleotides, etambutol oligonucleotides, and ID NO: 37-229.

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