RU2633507C2 - Method for molecular-genetic detection of tuberculosis microbacteria resistance to antitubercular preparations of second series (fluoroquinolones, aminoglycosides and capreomicin) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method for determination of mycobacteria tuberculosis resistance to antitubercular preparations of the second series - fluoroquinolones, aminoglycosides and capreomicin, sets of detection probes and amplification primers and method for confirmation of the clinical diagnosis of extensively drug-resistant tuberculosis. The method involves simultaneous detection of mutations in the tuberculosis mycobacteria DNA in a biological sample, leading to microorganisms resistance to antitubercular preparation of the second series in the sites of gyrA gene (codons 89, 90, 91, 94), gyrB gene (538, 539, 540), rrs gene (positions 1401, 514, 517) and the eis promoter region (positions -10, -12, -37), by real-time MPCR using primers for amplification and detection probes complementary to these gene sites.

EFFECT: simultaneous and rapid determination of tuberculosis mycobacteriua resistance to second-series preparations, and can be used in the diagnosis of extensively drug-resistant tuberculosis.

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Description

FIELD OF THE INVENTION

The proposed model relates to medicine, in particular to the field of treatment of tuberculosis, and relates to a method for detecting mutations based on real-time multiplex PCR technology for rapid determination of the resistance of tuberculosis mycobacteria (MBT) in biological samples to second-line anti-TB drugs, such as fluoroquinolones (ofloxacin, moxifloxacin) and reserve-type injectable anti-TB drugs (kanamycin, amikacin and capreomycin) (hereinafter referred to as second-line drugs).

BACKGROUND OF THE INVENTION

Tuberculosis is one of the most significant global health security concerns. Despite the general tendency towards a decrease in the incidence of tuberculosis, the share of tuberculosis in the structure of mortality of the population of Russia from infectious and parasitic diseases remains the main one, and amounts to about 56.0% according to 2012 data (Nechaeva O.B., Biragova OK 2013 Epidemic situation with tuberculosis in the Russian Federation [Electronic resource]. URL: http: //vestnik.mednet.ru/content/view/514/30/lang,ru/ (accessed 11/26/2015).

Recently, there has been a threat of the spread of an extremely dangerous form of tuberculosis - tuberculosis with resistance to anti-TB drugs (PTP), such as rifampicin and isoniazid, known as multidrug-resistant tuberculosis (MDR MDT) (WHO. Global Tuberculosis Control, 2011). The epidemiological situation is further exacerbated by the spread of extensively drug-resistant tuberculosis (XDR), i.e. a combination of MDR with resistance to at least one of the drugs of the fluoroquinolone series and one of the injection PTP of the reserve series (kanamycin, amikacin and capreomycin). More than 30,000 new XDR cases are registered worldwide each year. Moreover, there are data on cases of absolutely resistant tuberculosis, i.e. form resistant to all existing anti-TB drugs (Fattorini L., Migliori GB, Cassone A. 2007. Extensively drug-resistant (XDR) tuberculosis: an old and new threat. - Ann. Ist. Super. Sanita, 43 ( 4), p. 317-319; Haydel SE 2010. Extensively Drug-Resistant Tuberculosis: A Sign of the Times and an impetus for Antimicrobial Discovery. - NIH Public Access Author Manuscript Pharmaceuticals (Basel), V. 3 (7), p. 2268-2290).

The main reason for the deterioration of the epidemiological situation of TB-resistant tuberculosis is the wide geographical distribution of highly virulent and highly transmissible drug-resistant strains of M. Tuberculosis of Beijing genotypes (B. Lopez et al. 2003 A marked difference in pathogenesis and immune response induced by different Mycobacterium tuberculosis genotypes Clin Exp Immunol. 2003 Jul; 133 (l): 30-7; Narvskaya O.V., Mokrousov I.V., Limeshchenko E.V. et al. Molecular epidemiology of tuberculosis // BCG. - 2000. No. 7- 8. - P. 4-6; Narvskaya O.V., Mokrousov I.V., Limeshchenko E.V. et al. Molecular genetic characteristics ikobaktery tuberculosis isolated in the North-West region of Russia // Tuberculosis today: Problems and Prospects M., 2000. - S. 210-21)..

Tuberculosis with MDR and XDR is difficult to treat with existing anti-TB drugs; treatment requires long-term (18-24 months) complex and expensive treatment that causes serious side effects (Haydel SE Extensively Drug-Resistant Tuberculosis: A Sign of the Times and an Impetus for Antimicrobial Discovery Pharmaceuticals (Basel). 2010 Jul 1; 3 (7): 2268-2290). The set of drugs of choice in the case of drug-resistant tuberculosis is significantly limited, therefore, quick and accurate diagnosis of the sensitivity of the office to anti-TB drugs, in particular to reserve drugs, is especially relevant. In addition, XDR treatment for tuberculosis is extremely long and costly.

Thus, the problem of resistance of the Office to drugs poses a great threat to the fight against tuberculosis and remains a matter of serious concern for global health security.

Analysis of drug resistance of the office is mainly carried out by cultivation methods with antibiotics in a dense and / or liquid nutrient medium. The analysis of drug resistance of MBT using these methods is extremely long, and takes from 1 to 3 months from the start of the study of diagnostic material to the treatment of the patient. Moreover, the analysis of resistance to second-line drugs is possible only in an extremely limited number of laboratories.

The main drugs of choice in the treatment of MDR-MBT are fluoroquinolone drugs, such as ofloxacin, moxifloxacin, levofloxacin and others, which have shown high activity against Mycobacterium tuberculosis complex with MDR [Chan E.D et al. Treatment and outcome analysis of 205 patients with multidrug-resistant tuberculosis // AJRCCM. - 2004. - p. 26].

According to the literature, there have been cases of primary resistance of the office to drugs fluoroquinolone rad [Delgado MV, Telenti A. In: Selected PCR protocols for Emerging Infectious Diseases / Persing DH, ed., Washington, 1996], which emphasizes the relevance of timely determination of the resistance of the office to this group of drugs, especially before treatment.

The target of the action of fluoroquinolones in the office is the ATP-dependent type II DNA topoisomerase (DNA gyrase), which catalyzes the formation of DNA supercoiled [Blanchard J.S. Molecular mechanisms of drug resistance in Mycobacterium tuberculosis // Annu. Rev. Biochem. - 1996. - V. 65. - P. 215-239], and consists of two GyrA subunits and two GyrB subunits [Chen FJ, Lo HJ (2003) Molecular mechanisms of fluoroquinolone resistance. J Microbiol Immunol Infect 36: 1-9.]. Mutations in the region of the gyrA gene limited by codons 88 and 94, which determines the resistance of the office to fluoroquinolones (mainly ofloxacin and moxifloxacin) in 64% -79% of MBT strains of Beijing origin. The predominant mutations causing cross-resistance to fluoroquinolones are substitutions in the codon 94G and 90V of the gyrA gene [Maruri F et al. 2012 [Electronic resource]. URL: http: //www.ncbi.nlm.nih.gov/pubmed/22279180 (accessed 11/26/2015), Singh P et al. 2015 [Electronic resource]. URL: http: //www.ncbi.nlm.nih.gov/pubmed/25052485/ (accessed 11/26/2015); Willby M. et al. 2015 Correlation between GyrA mutations and ofloxacin, levofloxacin and moxifloxacin cross-resistance in Mycobacterium tuberculosis]. The gyrA gene also contains neutral polymorphism at codon 95. According to sequencing data, the substitution at codon 95 causes the coding of either serine (AGC) in M. tuberculosis H37Rv and H37Ra strains, or threonine (ACC) in M. tuberculosis Beiging, Erdman, M. bovis BCG , M. africanum. However, these substitutions are not associated with resistance to fluoroquinolones [Takiff N.E. et al. Cloning and nucleotide sequence of Mycobacterium tuberculosis gyrA and gyrB genes and detected of quinolone resistance mutations // Antimicrob. Agents a Chemotherapy. - 1994. - V. 38. - p. 773-780]. Therefore, the detection of this S95T polymorphism proposed in some diagnostics, for example, in biochips [Zimenkov D. et al. 2013 Detection of second-line drug resistance in Mycobacterium tuberculosis using oligonucleotide microarrays] may lead to false positive results.

Mutations in the gyrB gene are found in 5-10% of fluoroquinolone-resistant strains, and only three mutations in codons N538D, T539N, E540V determine the resistance of tuberculosis mycobacteria to all fluoroquinolones [13. Singh P et al. 2015, 16. S. Malik et al. 2012 [Electronic resource]. URL: http: //journals.plos.org/plosone/article? Id = 10.1371 / journal.pone.0039754 (accessed 11/26/2015)].

It is also known about the mechanism for the development of resistance of MBT to injectable anti-TB drugs (kanamycin, amikacin and capreomycin) through the occurrence of a mutation in the 16S-pPHK (rrs) ribosomal gene at positions 1401, 514 and 517 (16.S. Georghiou et al. 2012 Evaluation of Genetic Mutations Associated with Mycobacterium tuberculosis Resistance to Amikacin, Kanamycin and Capreomycin: A Systematic Review. PLoS One. 2012; 7 (3): e33275).

Replacing A1401G in the rrs gene is most common in kanamycin-resistant strains, and leads to the highest level of resistance to kanamycin with a minimum inhibitory concentration (MIC) of ≥80 μg / ml and cross-resistance to amikacin and capreomycin (17. Maus CE, Plikaytis BB, Shinnick TM, Molecular analysis of cross-resistance to capreomycin, kanamycin, amikacin, and viomycin in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2005 Aug; 49 (8): 3192-3197). This mutation is the most diagnostically significant, because was found in more than 84% of Beijing MBT strains resistant to kanamycin (KAN). In addition, the A1401G mutation also leads to cross-resistance to amikacin and capreomycin in approximately 50% resistant to AMA and ATS strains. [Electronic resource]. URL: (http://www.ncbi.nlm.nih.gov/pubmed/23948547 (accessed 11/26/2015). Mutations of resistance to kanamycin and amikacin at positions 514 and 517 of the rrs gene are more rare, and were mainly detected in MBT strains of non-Beijing origin.

The effect of mutations in the promoter region of the eis gene on the development of MBT resistance to low doses of kanamycin was shown by M.A. Zaunbrecher et al. in 2009 (Zaunbrecher MA, Sikes RD Jr, Metchock B et al. Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis. Proc Natl Acad Sci USA. 2009 Nov 24; 106 (47): 20004-20009 [20004-20009 [20004-20009] Electronic resource]. URL: http: //www.ncbi.nlm.nih.gov/pmc/articles/PMC2785282/ (accessed 11/26/2015). The eis gene encodes the Eis acetyltransferase, which specifically acetylates kanamycin and amikacin, but the rate of acetylation kanamycin is significantly higher, which explains the lack of cross-resistance to amikacin in strains with mutations in the promoter region of the eis gene. t to a multiple increase in its expression and, consequently, to the accumulation of the enzyme and the increase in its effect, i.e., ultimately, to the inactivation of the antibiotic.

The occurrence of mutations at positions -10, -12, and -14 of the eis promoter region in kanamycin-resistant strains is estimated in some studies to be no higher than 10% [M. Gicalo et al. 2012 [Electronic resource]. URL: http: //jac.oxfordjoumals.org/content/67/9/2107.long (accessed 11/26/2015)]. A mutation at position -37 of the eis gene was found in some MBT strains, however, the diagnostic significance of this mutation of resistance to kanamycin was not previously discussed [Casali N. et al. 2012]. According to our data, the -37 mutation of the eis gene occurs with a frequency of more than 8% only in kanamycin-resistant XDR strains of Beijing origin that do not carry other mutations in the eis gene or mutation 1401 of the rrs gene. Therefore, the -37 mutation of the eis gene is an independent marker of kanamycin resistance.

Thus, the detection of resistance mutations in the eis gene can increase the overall detectability of kanamycin resistance in XDR strains from the current 58-63% to 82-87%.

Determination of known, mainly, point mutations associated with resistance to fluoroquinolones and reserve drugs (kanamycin, amikacin, and capreomycin) using molecular genetic detection and analysis methods can provide rapid diagnostics of MBT resistance to PTP.

Existing molecular genetic methods for determining the resistance or sensitivity of MBTs to specific PTPs are based on the detection of known mutations in MBT genes by PCR amplification, in particular real-time PCR amplification. PCR amplification generates the required number of copies of the studied DNA, after which mutations are detected using one of such detection methods as: multiplex PCR analysis with hybridization probes, allele-specific PCR analysis, hybridization on chips or strips, analysis by melting curves, conformational analysis of single chain fragments in denaturing gradient gel electrophoresis, etc. (Victor T. C., Jordan AM Detection of mutation in drug resistance genes of M. tuberculosis by a dot-blot hibridization strategy // Int. J. Tubercle and Lung Disease. - 1999. - V. 2. - P. 346-349; 23. Ramaswamy S, Musser J. Molec ular genetic basis of antimicrobial agent resistance in M. tuberculosis // Tubercle and Lung Disease. - 1998 - V. 79 - P. 3-29). Now all of the above methods, with the exception of PCR analysis, along with the advantages have a common disadvantage, which consists in using them only for scientific research purposes and in highly qualified and equipped laboratories. For the same reason, the method of sequencing, including pyrosequencing, has not yet been introduced into the practice of molecular genetic diagnostics to determine mutations of resistance to PTP, due to its high cost and low productivity.

Molecular genetic methods based on PCR analysis and the corresponding test systems (Xpert, GenoType MTBDRplus / GenoType MTBDRsl, etc.) are successfully implemented in the laboratory diagnosis of tuberculosis, and are promising from the standpoint of high diagnostic sensitivity, high productivity (number of analyzes per formulation ), and in terms of performance and speed (several hours). However, in none of the modern test systems, the ability to detect mutations in the eis gene has been implemented to detect all mutations of resistance to kanamycin, amikacin, and capreomycin that are currently known. In addition, none of the proposed methods based on PCR amplification does not implement the option of detecting mutations in closely spaced codons 89, 90, 91 and 94 of the gyrA gene.

The prototype of the invention is a method for determining mutations in the rpoB gene, responsible for the resistance of MBT to rifampicin and isoniazid by multiplex PCR amplification using hybridization probes (L. Welebob, publication WO 2013132447 A1 dated 09/12/2013), where multiplex PCR is understood as simultaneous several reactions under the same temperature conditions with simultaneous amplification and detection of targets, in this case, nucleotide substitutions at specific positions of the MBT genes.

The prototype uses only oligonucleotide probes complementary to regions of the rpoB, katG genes and the promoter region of the inhA gene to detect mutations of resistance to first-line antibiotics, rifampicin and isoniazid. In the prototype there is no information on the principles of selection of probes for PCR detection of mutations of resistance to fluoroquinolones and injectable anti-TB drugs of the reserve series (kanamycin, amikacin and capreomycin). The prototype does not report all PCR reactions in the same temperature regime and effective hybridization of probes to matrices (amplicons).

Thus, there is a need for a simple method for determining the resistance of Mycobacterium tuberculosis to second-line drugs by detecting all known diagnostically significant mutations of resistance to these drugs.

SUMMARY OF THE INVENTION

The present invention relates to a method for determining the resistance of mycobacterium tuberculosis to second-line anti-TB drugs, characterized in that the method comprises the simultaneous detection of mutations in the DNA of mycobacteria of a biological sample, leading to microorganism resistance to second-line anti-TB drugs in the regions of the gyrA, gyrB, rrs and promoter genes eis regions by real-time multiplex polymerase chain reaction using primers for amplification and detecting rows that are complementary to portions of said genes gyrA, gyrB, rrs promoter region and eis, where the second-line drugs are fluoroquinolones, aminoglycosides and capreomycin.

In particular, fluoroquinolones are ofloxacin, moxifloxacin, levofloxacin, and aminoglycosides are, in particular, kanamycin and amikacin.

In a preferred embodiment of the invention, all reactions of detection of mutations of resistance to second-line anti-TB drugs are carried out in the same temperature regime of amplification

In a further embodiment of the invention, the biological sample is directly a material of a clinical sample or a pre-grown culture of microorganisms.

In a preferred embodiment, the clinical specimen is sputum, exudate, flush or broncho-alveolar lavage.

In yet another embodiment of the invention, the method is carried out for more than one biological sample.

According to a further embodiment of the invention, the gene regions are selected from the group comprising codons 89, 90, 91 and 94 of the gyrA gene, codons 539 and 540 of the gyrB gene, positions 1401, 514 and 517 of the rrs gene, positions -10, -12 and -37 of the promoter region eis gene or combinations thereof.

In another embodiment, the detection probes are 19-24 nucleotide oligonucleotide sequences labeled with fluorophores and quenchers used to detect mutations in codons 89, 90, 91 and 94 of the gyrA gene and in codons 538, 539 and 540 of the gyrB gene to detect mutations resistance to fluoroquinolones.

In a further embodiment, the detection probes are 19-22 nucleotide oligonucleotide sequences labeled with fluorophores and quenchers, used to detect mutations at positions -10, -12 and -37 of the promoter region of the eis gene to detect mutations in resistance to kanamycin, amikacin and / or capreomycin.

In yet another embodiment of the invention, said detection probes are 20-22 nucleotide oligonucleotide sequences used to detect mutations at positions 1401, 514 and 517 of the rrs gene to detect mutations of resistance to kanamycin, amikacin and / or capreomycin.

In a preferred embodiment, the probes include the nucleotide sequences shown in SEQ ID NOs: 7-14b 17-19, 26-31, or combinations thereof.

In a further preferred embodiment, the amplification primers include the nucleotide sequences shown in SEQ ID NOs: 1-6, 15 and 16, 16, 20-25, or combinations thereof.

The present invention also relates to the use of the method of the invention to confirm the clinical diagnosis of extensively drug-resistant tuberculosis.

Additionally, the present invention relates to a set of detection probes for implementing the method according to the invention, in which these probes include nucleotide sequences selected from those presented in SEQ ID NO: 7-14, 17-19, 26-31, or a combination thereof.

In addition, the present invention relates to a set of amplification primers for implementing the method according to the invention, in which these primers include nucleotide sequences selected from the group consisting of those presented in SEQ ID NOs: 1-6,15, 16, 20-25, or a combination thereof .

The present invention provides a method for the rapid and reliable detection of resistance to antituberculosis drugs of mutant forms of mycobacterium tuberculosis by simultaneously detecting mutations in the genes gyrA and gyrB, rrs and eis during multiplex PCR amplification with a set of hybridization probes, further analysis according to the curves of accumulation of specific products reactions, simple and unambiguous interpretation of the results.

The technical result of the present invention is the simultaneous and rapid determination of the resistance of mycobacterium tuberculosis of biological samples to fluoroquinolones and injectable anti-TB drugs of a backup series with high detectability of mutations in the DNA of biological samples (in particular, more than 90% of mutations of resistance to fluoroquinolones and more than 82% of mutations of resistance to kanamycin , amikacin and capreomycin in clinical sputum samples). It is also important that the determination of the resistance of tuberculosis mycobacteria can be carried out not only on DNA isolated from grown MBT cultures, but also isolated directly from clinical samples (sputum, bronchoalveolar lavage (BAL)) obtained from patients with various forms of tuberculosis.

Also, the technical result is the ability to quickly analyze large amounts of DNA samples of mycobacterium tuberculosis for the presence of genotypic resistance to both second-line fluoroquinolones and the reserve series (kanamycin, amikacin and capreomycin), in particular, up to sixty-eight samples per 96-well PCR -plash, as well as the possibility of simultaneously setting two reaction mixtures to determine the resistance to fluoroquinolones and injectable anti-TB drugs of the reserve series, which only ko is limited by the capacity of a 96-well PCR plate.

An additional technical result is the possibility of detecting mutations in closely located codons of the 89, 90, 91, and 94 regions of the gyrA gene to determine the resistance of MBT strains to fluoroquinolones by conducting multiplex PCR amplification using a minimum number of only two hybridization probes, complementary or one, or both chains of the analyzed DNA region, selected for hybridization in one temperature regime.

Also a technical result of the invention is the introduction into the method for determining the resistance of tuberculosis mycobacteria of an additional detection target corresponding to mutations at positions -10, -12 and -37 in the promoter region of the eis gene, which is a new target for detecting mutations of resistance to kanamycin, amikacin and / or capreomycin .

Additionally, the method for determining the resistance of tuberculosis mycobacteria according to the invention, using hybridization probes to both strands of the target DNA, is quite reproducible, low-cost, and allows the detection of mutations in both DNA fragments under study, which simplifies and speeds up the result.

The method according to the present invention is safe, sufficiently affordable to carry out in a specialized laboratory, and faster than the methods for determining stability known in the art (less than one day).

Another technical result of the invention is the simplification and minimization of the number of necessary sequential steps during PCR analysis in one test tube and thus reducing the possible intralaboratory contamination.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for determining the resistance of mycobacterium tuberculosis to second-line anti-TB drugs, characterized in that the method comprises the simultaneous detection of mutations in the DNA of mycobacteria of a biological sample, leading to microorganism resistance to second-line anti-TB drugs in the regions of the gyrA, gyrB, rrs and promoter genes eis regions by real-time multiplex polymerase chain reaction using primers for amplification and detecting rows that are complementary to portions of said genes gyrA, gyrB, rrs promoter region and eis, where the second-line drugs are fluoroquinolones, aminoglycosides and capreomycin.

Second-line anti-TB drugs are drugs that are used in tuberculosis patients who have tuberculosis mycobacteria resistance to first-line drugs. Second and reserve anti-TB drugs are widely known in the art and are, for example, fluoroquinolones, aminoglycosides and the capreomycin polypeptide. In particular, fluoroquinolones are ofloxacin, moxifloxacin, levofloxacin, and aminoglycosides are, in particular, kanamycin and amikacin.

The principle of the method according to the invention is based on the use of the process of multiplex PCR amplification of DNA, consisting in repeated cycles of temperature denaturation of one or more different DNA matrices, annealing of primers with complementary sequences and subsequent elongation of polynucleotide chains from these primers with Taq polymerase. Hybridization probes containing fluorescent labels and quenchers are introduced into the mixture for amplification, which allow detection of wild-type genetic variants in codons 89, 90, 91 and 94 of the gyrA gene, in codons 539 and 540 of the gyrB gene, at positions 1401, 514 and 517 of the rrs gene, at positions -10, -12 and -37 of the promoter region of the eis gene. The nucleotide composition, size and orientation of the probes are selected in such a way as to ensure hybridization of all probes with the DNA matrix at the same temperature conditions.

The absence of a genetic mutation (wild-type variant) is judged by the presence of an accumulation curve with high efficiency of fluorescence growth with an exponential growth of the curve of a specific product with a fluorescence growth efficiency of at least 90%. The absence of an accumulation curve or the low efficiency of the increase in fluorescence in the sample indicates the presence of a mutation of a certain type. Positive wild-type controls indicate detection by a set of wild-type variants and can eliminate false-positive results. To simultaneously detect the presence or absence of mutations, all PCR amplification reactions are carried out in the same temperature regime. Positive mutation controls allow the identification of MTB samples containing a certain type of mutation and indicate the absence of false negative results. Negative controls exclude cross-contamination of samples and non-specific reactions. In samples not containing MTB DNA, the result should be negative.

Thus, the present invention allows simultaneous PCR detection of the presence or absence of mutations of resistance to fluoroquinolones and injectable anti-TB drugs immediately after isolation of MBT DNA from a biological sample, while simultaneously determining the presence or absence of mutations in twelve positions of four genes, and to reduce the percentage of false negative results due to detection of additional mutations (in particular, mutations at positions 89, 91 in the gyrA gene, positions 539 or 540 in the gyrB gene, and position -37 in the promoter eis gene), and also allows you to reduce the cost of analysis and reduce the time it takes.

In a preferred embodiment of the invention, the resistance of mycobacterium tuberculosis to second-line anti-TB drugs is determined simultaneously in regions of genes selected from the group consisting of codons 89, 90, 91 and 94 of the gyrA gene, codons 539 and 540 of the gyrB gene, positions 1401, 514 and 517 of the rrs gene , positions -10, -12 and -37 of the promoter region of the eis gene or a combination thereof.

In the present invention, the biological sample is directly the material of a clinical sample or a pre-grown culture of microorganisms. In particular, the clinical specimen is sputum, exudate, flush or broncho-alveolar lavage.

The method according to the invention can be carried out for more than one biological sample, for example, for 45 or 68 samples, in fact, the number of samples per set is determined only by the capacity of a 96-well PCR machine and the possibility of simultaneously setting two reaction mixtures to determine resistance to fluoroquinolones and injectable anti-tuberculosis drugs reserve row (kanamycin, amikacin and capreomycin). Preferably, the number of samples per formulation is from a few to 43-45 (depending on the number of controls) for all mutations simultaneously (resistance to fluoroquinolones and kanamycin, amikacin and capreomycin) for 96-well PCR machines. In the case of posing only a mutation to fluoroquinolones or only to kanamycin, amikacin and capreomycin, the number of samples can reach 91.

The present invention also relates to a method in which all reactions of detection of mutations of resistance to second-line anti-TB drugs are carried out in the same temperature amplification mode.

As a result of varying the annealing temperatures of the primers and probes, the time of each cycle and the number of cycles, and the selection of the optimal Taq polymerase, the optimal amplification mode was selected, which is the same for both reaction mixtures for the detection of resistance to fluoroquinolones and injectable anti-tuberculosis drugs of the reserve series (kanamycin, amikacin and capreomycin). Primary denaturation and initialization conditions were selected depending on the choice of chemically modified Taq polymerase, such as AmpliTaqGold (Roche, US) or Taq DNA polymerase (MBI Fermentas), and were determined in the range of 6-12 min. at a temperature of 95 ° C-96 ° C. The annealing temperature of the primers was selected using the Primer 3 primer selection program (version 0.4.0) in the range 60 ° C-64 ° C, with an optimum value of 62 ° C.

The hybridization temperature of probespecific probes was selected in the range of 64 ° C-67 ° C, with a given optimal temperature of 66 ° C-67 ° C.

The elongation time was taken in the range of 20-30 seconds. at a temperature of 72 ° C, which was determined by the range of amplicon lengths of 130-220 bp

Also, due to the different bacterial load of the analyzed samples, and hence the different amount of the DNA template being introduced for PCR amplification, a scheme for setting up multiplex PCR amplification using preamplification in the amount of at least nine to fifteen cycles without reading the detection signals, and the subsequent 30 -38 cycles with reading of the detection signals at elevated annealing temperature (65 ° С-67 ° С).

Finally, the following optimal regimen of multiplex PCR amplification was chosen, which is common for both reaction mixtures for detecting resistance to fluoroquinolones and injectable anti-TB drugs of the reserve series (kanamycin, amikacin and capreomycin), which is as follows: 1st stage -95 ° C - 6-8 min; 2nd stage - 95 ° С - 15 sec, 62 ° С - 20 sec, 72 ° С - 25-30 sec (9 cycles); 3rd stage: 95 ° С - 15 sec, 66 ° С - 40-45 sec (30-35 cycles with reading).

In the method for determining the resistance of mycobacterium tuberculosis to second-line anti-TB drugs of the present invention, amplification primers and detection probes that are complementary to said regions of the gyrA, gyrB, rrs and eis genes are used. Hybridization probes can detect and differentiate both mutant and wild types of pathogens, as well as closely located mutations of resistance to fluoroquinolones. The probes can be complementary to regions of the gyrA, gyrB, rrs, and eis genes that do not contain a mutation, but can also contain mutations when they are used to confirm specific mutations. The proposed probes containing mutations are labeled with a different fluorophore, unlike probes that do not include a mutation in their sequence, i.e. wild type, in cases where it is important to check and confirm the presence of certain mutations, such as the main mutation of resistance to aminoglycosides and capreomycin a1401g rrs or c-12t promoter region of the eis gene.

These primers are used to amplify short specific fragments, amplicons of the gyrA and gyrB genes (in the case of determination of resistance to fluoroquinolones) and the rrs gene and the promoter region of the eis gene (in the case of determination of resistance to kanamycin, amikacin and capreomycin) for specific PCR detection of sites of these genes with subsequent detection of the presence or absence of mutations of resistance to these antibiotics in the DNA samples of mycobacterium tuberculosis.

Detection probes are modified oligonucleotides, usually 18-27 nucleotides long, that carry a fluorophore sewn to the 5'-end of the probe and a quencher sewn to the 3'-end. The fluorescence of the probe, and hence the detection of the PCR amplification product, occurs during the PCR reaction after hybridization of the probe with complementary DNA regions, while the fluorophore emits a signal, and the quencher is spatially separated from the fluorophore only during hybridization and as a result of using Taq polymerase with 5 ' exonuclease activity.

In the method according to the present invention, said detection probes can be 19-24 nucleotide oligonucleotide sequences used to detect mutations in codons 89, 90, 91 and 94 of the gyrA gene and in codons 538, 539 and 540 of the gyrB gene to detect fluoroquinolone resistance mutations .

In addition, in the method according to the present invention, these detection probes can be oligonucleotide sequences 19-22 nucleotides long, used to detect mutations at positions -10, -12 and -37 of the promoter region of the eis gene to detect mutations of resistance to kanamycin, amikacin and / or capreomycin.

In one embodiment of the invention, said detection probes are 20-22 nucleotide oligonucleotide sequences used to detect mutations at positions 1401, 514 and 517 of the rrs gene to detect mutations in resistance to kanamycin, amikacin and / or capreomycin.

These oligonucleotide sequences can be labeled with fluorophores and quenchers.

To identify mutations in the gyrA and gyrB genes of mycobacterium tuberculosis, primer pairs and hybridization probes were selected to amplify specific fragments of the gyrA and gyrB genes. A PCR amplification program was selected for PCR reactions and probe hybridization in one temperature mode, and a method was proposed for differentiating closely spaced single nucleotide substitutions with two probes in the four codons 89, 90, 91, and 94 of the gyrA gene.

To solve the problem of detection and differentiation of nearby mutations in the gyrA and gyrB genes, conditions were first developed for each of the stages of the method:

For the synthesis of MBT DNA of the gyrA gene, primers were selected using the Primer3 and BioEdit computer programs:

Primers were selected for amplification of the region of the gyrA gene, spanning the region with codons 88-95, not more than 140 bp:

SEQ ID NO 1: Straight 5'-CCGAGACCATGGGCAACTAC-3 '

SEQ ID NO 2: Inverse 5'-GCTTCGGTGTACCTCATCG-3 '

or

SEQ ID NO 3: Straight 5'-GAGACCATGGGCAACTACCA-3 '

SEQ ID NO 4: Inverse 5'-ATGACCCACCGGCGGCGATG-3 '

To amplify the gyrB gene region, primers were selected that flank the gene region between codons 516 and 550:

SEQ ID NO 5: grB PRIMER 5'-TTCGATGTTCCAGGCGATAC-3 '

SEQ ID NO 6: grB PRIMER 5'-TCTTGTGGTAGCGCAGCTTG-3 '

To ensure species specificity of the selected primers, a comparative analysis of the nucleotide sequences was carried out with the GenBank database using the BLAST program and e-PCR. It was found that there are no cross-reactions when using selected primers with DNA of other types of microorganisms and humans.

Next, hybridization probes were selected to perform multiplex PCR amplification together with the selected primers to detect mutations in codons 90-94 of the gyrA gene region. The probes were withdrawn both in the forward and reverse directions to ensure hybridization in the temperature range: 64-67 ° C.

SEQ ID NO 7: 94 FL1 -5'-ACGCGTCGATCTACGACACCCTG-BHQ1

SEQ ID NO 8: 94-c FL1 -5'-AGGGTGTCGTAGATCGACGCG-BHQ1

In order to avoid spatial complications during hybridization of closely located probes, a more preferable option was chosen for positions 91 and 94, in which probes are used that are complementary to the (-) DNA strands of the template at positions 89 and 90.

Probes for specific hybridization with codons 89-91 of the gyrA gene region, optimized for hybridization temperature in the same temperature range of 64-67 ° C, the following are proposed:

SEQ ID NO 9: 90-91-s FL2-5'-TAGATCGACGCGTCGCCGT-3'-BHQ2

SEQ ID NO 10: 91-90 FL2-5'-ACGGCGACGCGTCGATCTACG-3'-BHQ2

The principle of differential detection of major mutations in the gyrA gene is realized in such a way that, for example, a mutation in the 94 codon of the gyrA gene is detected by the absence of an accumulation curve through one fluorophore recognition channel, for example, FAM with the simultaneous presence of an accumulation curve through another fluorophore recognition channel, for example, ROX with the efficiency of the increase in fluorescence close to 100%. Mutations in codon 91 of the gyrA gene are detected by the simultaneous absence of accumulation curves on both fluorophore recognition channels.

The absence of mutations in codons 89 and 90 of the gyrA gene is determined by the presence of accumulation curves for both channels, for example, FAM and ROX, with a fluorescence gain of close to 100%.

Mutations in codon 94 are detected by the absence of an accumulation curve on the FAM channel with a fluorescence gain of less than 10%.

Mutations in codons 89 and 90 of the gyrA gene are detected by the presence of an accumulation curve along one channel, for example, FAM, with fluorescence growth efficiencies close to 100% and less than 40%, respectively, while the absence of a curve is characteristic for another mutation on the other channel, for example, ROX accumulation. The presence of polymorphism in codon 95 is also determined by hybridization with a probe complementary to region with codon 94, and is detected by the accumulation curve with a fluorescence growth efficiency close to 70%, which does not affect the ability to differentiate mutant and wild forms from codon 94.

The interpretation of mutation detection results in codons 89, 90, 91, and 94 of the gyrA gene according to the amplitude of the detection signal for two probes is presented in Table 1.

Thus, our proposed option for detecting mutations in closely located codons of the 89, 90, 91, and 94 regions of the gyrA gene is provided by multiplex PCR amplification using only two hybridization probes, complementary to one or both chains of the analyzed DNA region, but chosen for hybridization in one temperature range 64-67 ° С.

In addition, the approach used allows one to avoid detection of nearby single-nucleotide polymorphism in codon 95, thereby only detecting fluoroquinolone resistance mutations.

A hybridization probe specific for the gyrB gene site at positions 538-540, both direct and complementary, from the following group was also selected:

SEQ ID NO 11: FL3 -5'-TAAAGAACACCGAAGTTCAGGCG-3-BHQ1

SEQ ID NO 12: FL3-5'-AAAGAACAACGAAGTTCAGGCG-3-BHQ1

SEQ ID NO 13: FL3-c-5'-CGCCTGAACTTCGGTGTTCTTTA-3-BHQ1

SEQ ID NO 14: FL3-c-5'-CGCCTGAACTTCGGTGTTCTTT-3-BHQ1

Preferred is SEQ ID NO 11, because provides the best detection of possible substitutions in any of the codons 538-540.

The next step was the development of a method for detecting resistance mutations in mycobacterium tuberculosis against injectable anti-tuberculosis drugs of the reserve series (kanamycin, amikacin and capreomycin).

Our sequencing data for the determination of mutations in the resistance of MBT to kanamycin, amikacin, and capreomycin, as well as available literature data indicate the need to search for mutations both at positions 1401, 514, 517 of the rrs gene, and in the promoter region of the eis gene at positions -10, -12, and -37.

The low GC composition and the length of the promoter region of the eis gene allow the use of multiplex PCR amplification with the simultaneous use of two hybridization probes against the site with positions -10 / -12 and against position -37 to detect mutations of interest to us. Accordingly, to amplify a specific region of the promoter region of the eis gene, pairs of primers of the following composition were selected:

SEQ ID NO 15: Direct 5'-atcgcgtgatcctttgccag-3 '

SEQ ID NO 16: Reverse 5'-gcggccagtaggaacatcc-3 '

The species specificity of the selected primers was determined using a comparative analysis of the nucleotide sequences from the GenBank database using the BLAST program and e-PCR. It was found that there are no cross-reactions when using selected primers with DNA of other types of microorganisms and humans.

Next, hybridization probes were selected to perform multiplex PCR amplification together with the selected primers to detect mutations at positions -10 and -12, as well as at position -37 of the promoter region of the eis gene. To differentiate the mutations in positions -10 and -12, the following probes with a hybridization temperature range of 65-67 ° C were selected:

SEQ ID NO 17: eis-10 FL1 - catatgccacagtcggattctgtgac-BHQ1

SEQ ID NO 18: eis-12 mutant FL2-ATATGCCACAATCGGATTCTGTGACTGTG-BHQ1

In this case, the probe SEQ ID NO 15, labeled with another fluorophore, allows you to detect only a mutant form with a mutation at position -12 of the promoter region of the eis gene in biological samples of mycobacterium tuberculosis.

A hybridization probe recognizing the wild-type variant at position -37 was selected as follows:

SEQ ID NO 19: eis-37 FL3- tcgtcgtaatattcacgtgcacgt-BHQ1

The method of the invention can use standard amplification primers and detection probes that are known in the art (Suzuki YC et al. Detection of kanamycin-resistant Mycobacterium tuberculosis by identifying mutations in the 16S rRNA gene. // J. Clin. Microbiol. - 1998. - v. 36. - p. 1220-1225) and are used to determine the resistance to reserve drugs for the treatment of tuberculosis by the presence or absence of mutations in the rrs gene. But to achieve the best detectability, we created primers and probes, which are described in more detail below.

To identify known specific mutations in the resistance of tuberculosis mycobacteria to kanamycin / amikacin and capreomycin at position 1401 of the rrs gene, as well as mutations in 514 and 517 associated with kanamycin / amikacin resistance, primer pairs and hybridization probes were selected to amplify specific fragments flanking these mutations of genes.

To ensure species-specificity of the selected primers for amplification of highly conserved regions of the rrs gene where mutations are localized, a preliminary analysis of nucleotide sequences 80-110 bp in length located at the left and right of the mutations was performed with the GenBank database using the BLAST program and e-PCR. Primers were selected to ensure the absence of cross-reactions with DNA of other types of microorganisms and humans. Primers for amplification of the plot of the rrs gene, spanning plot 1390-1490:

SEQ ID NO 20: Direct 5'-CGCGAGGTTAAGCGAATCCTTA-3 '

SEQ ID NO 21: Direct 5'-GCGAGGTTAAGCGAATCCTTA-3 '

SEQ ID NO 22: Inverse 5'-TCGACAGCTCCCTCCCGA-3 '

SEQ ID NO 23: Inverse 5'-CGACAGCTCCCTCCCGAG-3 '

For PCR amplification of the rrs gene region in the annealing temperature range of 61-64 ° C, a pair of primers SEQ ID NO 21 and SEQ ID NO 22 turned out to be preferable.

Primers for the rrs gene region flanking mutations at positions 514 and 517 are not in the region of high conservatism. The species specificity of the selected primers was also determined using a comparative analysis of the nucleotide sequences from the GenBank database using the BLAST program and e-PCR. It was found that there are no cross-reactions when using selected primers with DNA of other types of microorganisms and humans.

Accordingly, in order to amplify a specific region of the rrs gene, spanning positions 514 and 517, a pair of primers of the following composition were selected in the same temperature range 61-64 ° C:

SEQ ID NO 24: Direct 5'-cgaaggtccgggttctctcc-3 '

SEQ ID NO 25: Reverse 5'-cgcccagtaattccggaca-3 '

Next, hybridization probes were selected for the multiplex PCR amplification with the selected primers to detect mutations in regions 514 and 517 of the rrs gene. The probes were withdrawn both in the forward and reverse directions to ensure hybridization in the temperature range: 64-67 ° C.

SEQ ID NO 26: 514 / 517for1 FL2-tgccagcAgcCgcggtaat-BHQ2

SEQ ID NO 27: 514 / 517for2 FL2-ccagcAgcCgcggtaatacgt-BHQ2

SEQ ID NO 28: 514 / 517rev1 L2-ATTACCGCGGCTGCTGGCA-BHQ2

SEQ ID NO 29: 514 / 517rev2 FL2-ATTACCGCGGCTGCTGGCA-BHQ2

The preferred option was the probe SEQ ID NO 28, because provides better detection during hybridization with possible replacements in any of the a514c and c517t positions.

To detect mutations at position 1401 and an extremely rare mutation at position 1402, the following probes with a hybridization temperature range of 64-67 ° C were selected:

SEQ ID NO 30: rrs 1401 FL1-ACCGCCCGTCACGTCATGAA-BHQ1

SEQ ID NO 31: rrs 1401 mutant FL2-ACCGCCCGTCGCGTCATGAA-BHQ1

In this case, the probe SEQ ID NO 31, labeled with a different fluorophore, allows only the mutant form with the a1401g mutation to be detected in biological samples of tuberculosis mycobacteria.

Thus, primers were selected, specific hybridization probes for amplification (61-63 ° C) and detection in the same temperature regime (64-67 ° C) of mutations of resistance to fluoroquinolones and kanamycin, amikacin and capreomycin.

The PCR conditions for all targets are as follows: 1st stage - 95 ° - 6-8 min; 2nd stage - 95 ° - 15 sec, 62 ° - 20 sec, 72 ° - 25 sec (9 cycles); 3rd stage: 95 ° - 15 sec, 66-67 ° - 40 sec. (30-35 cycles in read mode).

To select the optimal conditions for multiplex PCR amplification, the composition of the reaction buffer was also selected in which the important components are the ratio of the number of introduced primers and probes, the concentration of triphosphates, and the concentration of magnesium ions. In the reaction volume of the multiplex mixture is 22-24 μl containing 10 mM Tris-HCl pH 8.8-9.0; 50-70 mM KCl; 0.5% Tween 20, 1.8-3.5 mM MgCl ₂ , 3.0-4.0 units Taq polymerase, 200 μM of each nucleoside triphosphate, 5-9 pmol of each oligonucleotide primer and 5-10 pmol of probes contributed 12 μl of the DNA sample.

The technique used by us included the following steps: growing MBT cultures in a WASTES selective medium with antibiotics, thereby determining resistance or sensitivity, isolating DNA from phenotypically characterized cultures, staging the MPCR and checking for mutations by sequencing.

DNA was isolated from MBT cultures obtained on Middlebrook 7H9 liquid nutrient medium in the WASTEC MGIT 960 automated system (Becton Dickinson, United States), by heating at 95 ° С for 20 min in TE buffer with 1% Triton (pH 8.8).

To amplify regions of the rrs and eis genes, an optimized 2X reaction mixture similar to that proposed for gyrA and gyrB was used. PCR amplification was performed in a total volume of up to 24 μl, with the amount of introduced DNA up to 12 μl.

The temperature regime of amplification was chosen uniform for both gyrA and gyrB: 1st stage - 95 ° - 6-8 min; 2nd stage - 95 ° - 15 sec, 62 ° - 20 sec, 72 ° - 25 sec (9 cycles); 3rd stage: 95 ° - 15 sec, 66 ° - 40 sec (35 cycles - in read mode).

The present invention also relates to a set of detection probes for implementing the method according to the invention, wherein said probes include nucleotide sequences selected from the group consisting of those presented in SEQ ID NOs: 7-14, 17-19, 26-31, or a combination thereof.

In addition, the present invention relates to a set of amplification primers for implementing the method according to any one of paragraphs. 1-10, in which these primers include nucleotide sequences selected from the group consisting of those presented in SEQ ID NO: 1-6, 15, 16, 20-25, or a combination thereof.

These kits may also include instructions for use in a method for determining the resistance of Mycobacterium tuberculosis to second and reserve row anti-TB drugs.

Example 1

Detection of fluoroquinolone resistance mutations (ofloxacin, moxifloxacin, levofloxacin) in the genes gyrA and gyrB on isolates isolated from clinical samples.

A PCR analysis of samples from 39 isolates, monocultures isolated from clinical samples characterized by phenotypic cultivation in WASTES medium with a concentration of ofloxacin (OFX) and levofloxacin (LVX) at 2 mg / l and moxifloxacin (MFX) 0.25 mg / l Samples resistant and sensitive to these antibiotics were verified by sequencing the genes gyrA and gyrB. In all cases, the results of DNA analysis coincided with phenotypic data.

PCR analysis was performed using 10-12 μl of purified DNA, isolated from each isolate by magnetic particle sorption with a commercial Promega kit, which was added to a 2-fold reaction mixture (final volume 24 μl) containing 5-6 pmol of each of the primers SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 6, 5-10 pmol of each probe SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 14, 2.5 mM MgCl ₂ , 3 units Taq DNA polymerase (MBI Fermentas), and 200 microM each of dNTP. PCR reactions were carried out on a DT-96 thermocycler (DNA technology, RF) under identical conditions according to the protocol with preamplification: 1st stage - 95 ° - 6 min; 2nd stage - 95 ° - 15 sec, 62 ° - 20 sec, 72 ° - 25 sec (9 cycles); 3rd stage: 95 ° - 15 sec, 66 ° - 40 sec (30 cycles with reading).

The results of the detection of mutations of resistance to fluoroquinolones by the proposed method and verification by sequencing of some isolates are presented in Table 2. Sequencing revealed a polymorphism with the replacement of the S95T amino acid in both fluoroquinolone-resistant and sensitive samples. However, during PCR detection by the proposed method, this polymorphism did not interfere with real closely located mutations in codon 94.

Example 2

Detection of mutations of resistance to injectable anti-TB drugs of the reserve series (kanamycin, amikacin and capreomycin) in the rrs and eis genes on isolates isolated from clinical samples.

Analysis of samples from 39 isolates, monocultures isolated from clinical samples characterized by phenotypic cultivation in WASTES medium with a concentration of kanamycin, amikacin and capreomycin of 30 mg / l was carried out using the proposed MPCR method with subsequent verification of the results by sequencing. In all cases, the results of DNA analysis from the same strains coincided with phenotypic data.

PCR analysis was carried out using 10-12 μl of purified DNA isolated from isolates by magnetic sorption using a commercial Promega kit, which was added to a 2-fold reaction mixture (final volume 24 μl) containing 5-6 pmol of each of the primers SEQ ID NO 15 and SEQ ID NO 16, SEQ ID NO 21 and SEQ ID NO 22, SEQ ID NO 24 and SEQ ID NO 25, 5-10 pmol of each probe SEQ ID NO 17 and SEQ ID NO 19, SEQ ID NO 28 and SEQ ID NO 30, 2.5 mm MgCl ₂ , 3 units Taq DNA polymerase (MBI Fermentas), and 200 microM each of dNTP. PCR reactions were carried out on a DT-96 amplifier (DNA-technology, RF) under the same conditions according to protocol c and were carried out under identical conditions according to the protocol with preamplification: 1st stage - 95 ° - 6 min; 2nd stage - 95 ° - 15 sec, 62 ° - 20 sec, 72 ° - 25 sec (9 cycles); 3rd stage: 95 ° - 15 sec, 66 ° - 40 sec (30 cycles with reading).

The results of the detection of resistance to kanamycin, amikacin and capreomycin by three cultivation methods, the proposed method of MPCR and sequencing are presented in table 2. As a result, it is shown that the mutation resistance to aminoglycosides and capreomycin in the rrs gene are detected in the isolates of samples with the XDR phenotype, such as in position 1401, mutations of resistance only to aminoglycosides at positions 514c and 517t of the rrs gene, as well as mutations in the promoter region of the eis gene at positions -10 and -37. Mutations detected at the -12 position of the eis gene also cause capreomycin resistance, which is of diagnostic significance.

Example 3

Detection of mutations in the resistance of tuberculosis mycobacteria to fluoroquinolones and injectable anti-TB drugs in isolates isolated from clinical sputum samples by the proposed method for determining the diagnostic effectiveness (sensitivity and specificity) of a prototype diagnostic test system

Analysis of samples from 68 isolates, monocultures, isolated from clinical samples, was carried out by the proposed method for the simultaneous formulation of MPCR detection of mutations of resistance to fluoroquinolones (ofloxacin) and kanamycin, amikacin and capreomycin. All samples were previously phenotypically determined by cultivation in WASTES medium, as well as sequencing, as 34 with extensive drug resistance and 34 sensitive to second-line antibiotics. In all cases, the results of DNA sequencing from the same strains coincided with phenotypic data.

PCR analysis was performed using 10-12 μl of purified DNA isolated from isolates by magnetic sorption using a commercial Promega kit, which was added to a 2-fold reaction mixture (final volume 24 μl) containing 5-6 pmol of each of the primers listed in Examples 1 and 2, 5-10 pmol of each of the probes listed in Examples 1 and 2, 2.5 mm MgCl ₂ , 3 units. Taq DNA polymerase (MBI Fermentas), and 200 microM each of dNTP. All PCR reactions are carried out under identical conditions according to the protocol with preamplification: 1st stage - 95 ° - 6 min; 2nd stage - 95 ° - 15 sec, 62 ° - 20 sec, 72 ° - 25 sec (9 cycles); 3rd stage: 95 ° - 15 sec, 66 ° - 40 sec (30 cycles with reading).

According to the results of the detection of mutations of resistance to injectable anti-tuberculosis drugs kanamycin, amikacin and capreomycin (SL) and fluoroquinolone ofloxacin (FQ) by the proposed method and verification by sequencing, diagnostic characteristics (sensitivity and specificity) were determined, as presented in table 4. Only three of 34 XDR samples, no fluoroquinolone resistance mutations were detected at positions 89, 90, 91 and 94 in the gyrA gene, 539 and 540 in the gyrB gene, which was confirmed by complete sequencing reading frames of these genes. In five of 34 XDR samples, no known mutations of resistance to kanamycin, amikacin, and capreomycin were detected, as well as mutations proposed in the promoter region of the eis gene, which is also confirmed by sequencing of the rrs and eis genes.

The diagnostic sensitivity for the detection of ofloxacin resistance was 91.2% (31/34), while the diagnostic sensitivity for injectable anti-TB drugs was 82.4% (28/34), which is higher than the existing commercial test systems, such as GenoType MTBDR / j / ms / GenoType MTBDRs /, due to the lack of detection of mutations in the promoter region of the eis gene. The contribution of mutations in the promoter region of the eis gene at positions –10, –12, and –37 was about 39% among all XDR samples, if we take into account that mutations c517t and a514c of the rrs gene were also found in some of them. Only mutations at positions –10, –12, and –37 of the eis gene were found in 23.5% (8/34) of all XDR specimens with resistance to kanamycin and amikacin, which apparently reflects the diagnostic significance of these mutations.

Diagnostic specificity was 100%, because no mutations were detected among sensitive samples.

Claims (11)

1. A method for determining the resistance of Mycobacterium tuberculosis to second-line anti-TB drugs, where the method includes the simultaneous detection of mutations in the DNA of Mycobacterium tuberculosis biological sample, leading to the resistance of microorganisms to anti-TB drugs of the second row in the gene regions gyrA, gyrB, rrs and the promoter region of eis by multiplex real-time polymerase chain reaction using amplification primers and detection probes that are complementary to the indicated sections of the gyrA, gyrB, rrs genes and the eis promoter region, where the second-line anti-TB drugs are fluoroquinolones, aminoglycosides and capreomycin, where these sections of the gyrA gene are selected from the group comprising codons 89, 90, 91, 94 or combinations thereof, these sections the gyrB gene is selected from the group comprising codons 538, 539, 540 or a combination thereof, the indicated regions of the rrs gene are selected from the group comprising the positions 1401, 514, 517, or combinations thereof, and these regions of the eis promoter region are selected from the group comprising the positions -10 , -12, -37 or their comb initiation. 2. The method of claim 1, wherein the aminoglycosides are kanamycin and amikacin, the fluoroquinolones are ofloxacin, moxifloxacin, levofloxacin. 3. The method according to p. 1, where all the reactions of detection of mutations of resistance to second-line anti-TB drugs are carried out in the same temperature regime of amplification 4. The method according to p. 1, characterized in that said biological sample is directly a material of a clinical sample or a pre-grown culture of microorganisms. 5. The method according to p. 1, characterized in that the method is carried out for more than one biological sample. 6. The method according to any one of paragraphs. 1-5, where these detection probes are oligonucleotide sequences of 19-24 nucleotides in length used to detect mutations in codons 89, 90, 91 and 94 of the gyrA gene and in codons 538, 539 and 540 of the gyrB gene to detect mutations of resistance to fluoroquinolones, oligonucleotide sequences 19-22 nucleotides long used to detect mutations at positions -10, -12 and -37 of the promoter region of the eis gene to detect mutations of resistance to kanamycin, amikacin and / or capreomycin, and oligonucleotide sequences 20-22 nucleotides long, approx nyaemye for detecting mutations in positions 1401, 514 and 517 rrs gene to identify mutations of resistance to kanamycin, amikacin and / or capreomycin. 7. The method according to p. 6, where these probes include nucleotide sequences selected from those presented in SEQ ID NO: 7-14, 17-19, 26-31, or a combination thereof. 8. The method according to any one of paragraphs. 1-5, where these primers for amplification include nucleotide sequences selected from those presented in SEQ ID NO: 1-6, 15, 16, 20-25, or combinations thereof. 9. A set of detection probes for implementing the method according to any one of paragraphs. 1-8, in which these probes include nucleotide sequences selected from the group consisting of those presented in SEQ ID NO: 7-14, 17-19, 26-31, or a combination thereof. 10. A set of amplification primers for implementing the method according to any one of paragraphs. 1-8, in which these primers include nucleotide sequences selected from the group consisting of those presented in SEQ ID NO: 1-6, 15, 16, 20-25, or a combination thereof. 11. The application of the method according to any one of paragraphs. 1-8 to confirm the clinical diagnosis of extensively drug-resistant tuberculosis.