RU2175015C1 - Method of assay of single base exchanges in mycobacterium dna, method of diagnosis of resistance of mycobacterium to rifampicin, biochip for realization of these methods - Google Patents

Abstract

FIELD: molecular biology, microbiology, medicine. SUBSTANCE: method involves amplification of gene rpoB fragment to obtain a single-stranded fluorescently labeled product by method of polymerase chain reaction. Biochip for assay of rifampicin- -resistant mycobacterium is prepared and hybridization of amplified labeled product on biochip is performed. Results of hybridization are recorded and explained their by comparison of fluorescence signals obtained in completed and uncompleted hybridization. Biochip is micromatrix with cells where oligonucleotide set is immobilized and their sequences are given in description of the invention. Upper row of biochip has oligonucleotides that are complementary to DNA sequence of wild type. Corresponding column under each cell has oligonucleotides that are complementary to DNA sequences of mutant type responsible for exchange of a single amino acid. Diagnosis of resistance of mycobacterium to rifampicin is carried out by determination of single nucleotide exchanges in pathogen DNA. Invention ensures to perform analysis from clinical sample directly, to assay some mutations simultaneously. EFFECT: improved method of assay, decreased cost and time of analysis. 11 cl, 6 dwg, 2 tbl, 3 ex

Description

Technical field
The invention relates to molecular biology, microbiology and medicine and considers a method for determining the sensitivity of tuberculosis mycobacteria to rifampicin directly in a clinical sample using a differentiating biochip. The invention also includes methods for creating a differentiating oligonucleotide biochip, a methodology for preparing a clinical sample for the hybridization procedure, registration and interpretation of the results.

State of the art
The following methods are used to detect point mutations:
I. Extent of the chain labeled with dideoxyribonucleoside triphosphate (SNP-single nucleotide polymorphism);
Dubiley S., Kirillov E. and A. Mirzabekov. 1999. Polymorphism analysis and gene detection by minisequencing on an array of gel-immobilized primers. Nucleic Acids Research, Vol. 27, No. l8 (el9)
Marth GT, Korf I, Yandell MD et al. 1999. A general approach to single-nucleotide polymorphism discovery. Nat Genet., Dec; 23 (4): 452-456.

II. Allele-specific PCR (Allele specific PCR);
De los Monteras LEE, JCGalan, M. Gutierrez, S. Samper, JFGMarin, C. Martin, L. Doninguez, L. de Rafael, F.Baquero, E. Gomez-Mampaso, and J.Blazquez. 1998. Allele-Specific PCR Method Based on pncA and oxyR Sequences for Distinguishing Mycobacterium bovis from Mycobacterium tuberculosis: Intraspecific M. bovis pncA Sequence Polymorphism. J. Clin. Microbiol. 36 :, 239-242.

III. Study of fragment polymorphism after restriction (RFLP-restriction fragment length polymorphism);
PCR-RFLP Detection of point Mutations in the Catalase-Peroxidase Gene (katG) of Mycobacterium tuberculosis Associated with Isoniazid Resistance. In: PCR Protocols for Emerging Infectious Diseases (DHPersing, Ed.) (1996) ASM Press, Washington, pp. 144-149.

IV. Analysis of conformational polymorphism of single and double stranded DNA (SSCP and DSCP- single- (double) -strand conformation polymorphism);
Delgado MB and A.Telenti. Detection of Fluoroquinolone Resistance Mutations in Mycobacterium tuberculosis. In: PCR Protocols for Emerging Infectious Diseases (DH Persing, Ed.) (1996) ASM Press, Washington, pp. 138-143.

 Pretorius G.S., P.D. van Helden, F. Sirgel, K. D. Eisenach and T. C. Victor. 1995. Mutations in katG Gene Sequences in Isoniazid-Resistant Clinical Isolates of Mycobacterium tuberculosis Are Rare. Antimicrob. Agents Chemother., 39, 10, 2276-2281.

V. Hybridization on microarrays (Hybridization on microarray);
Gingeras TR, G. Ghandour, E. Wang, A. Verno, PMSmall, F. Drobniewski, D. Alland, E. Desmond, M. Holodniy, and J. Drenkow. 1998. Simultaneous Genotyping and Species Identification Using Hybridization Pattern Recognition Analysis of Generic Mycobacterium DNA Arrays. Genome Res., 8: 435-448.

 Troesch A., H. Nguyen, C. G. Miyada, S. Desvarenne, T. R. Gingeras, P. M. Kaplan., P. Cross, and C. Mabilat. 1999. Mycobacterium Species Identification and Rifampicin Resistance Testing with High-Density DNA Probe Arrays. J. Clin. Microbiol., 37: 49-55.

 VI. Sequencing Kapur, V., L.-L. Li, S. lordanescu, M. R. Hamrick, A. Wanger, B. N. Kre-iswirth, and J. M. Musser. 1994. Characterization by automated DNA sequencing of mutations in the gene (rpoB) encoding the RNA polymerase b subunit in rifampin-resistant Mycobacterium tuberculosis strains from New York City and Texas. J. Clin. Microbiol. 32: 1095-1098.

 Rys P. N. and T. A. Felmlee. Detection of Rifampicin Resistance Mutations in Mycobacterium tuberculosis by Dideoxy Fingerprinting. In: PCR Protocols for Emerging Infectious Diseases (D.H. Persing, Ed.) (1996) ASM Press, Washington, pp. 112-121.

VII. Dideoxyfingerprinting (ddE-method);
Rys PN and TAFelmlee. Detection of Rifampicin Resistance Mutations in Mycobacterium tuberculosis by Dideoxy Fingerprinting. In: PCR Protocols for Emerging Infectious Diseases (DHPersing, Ed.) (1996) ASM Press, Washington, pp. 112-121.

Viii. PCR heteroduplex analysis (PCR-heteroduplex analysis);
Williams DL, CWLimbers, L. Spring, S. Jayachandra, and TPGillis. PCR-Heteroduplex Detection of Rifampicin-Resistant Mycobacterium tuberculosis. In: PCR Protocols for Emerging Infectious Diseases (DHPersing, Ed.) (1996) ASM Press, Washington, pp. 122-129.

IX. Imperfect RNA duplex method (RNA mismatch analysis);
Dracopoli, NC Ed. "Detection of Mutations by RNase Cleavage." Current Protocols in Human Genetics. 1998. John Wiley & Sons, Inc.

 Marth GT, Korf I, Yandell MD et al. 1999. A general approach to single-nucleotide polymorphism discovery. Nat Genet., Dec; 23 (4): 452-456.

X. Method of structurally specific splitting (Structure-specific endonuclease cleavage);
Brow MAD, MCOldenburg, V. Lyamichev, LM Heisler, N. Lyamicheva, JG Hall, NJEagan, DMOlive, LMSmith, L. Fors, and JEDahlberg. 1996. Differentiation of Bacterial 16S rRNA Genes and Intergenic Regions and Mycobacterium tuberculosis katG Genes by Structure-Specific Endonuclease Cleavage. J. Clin. Microbiol., 34: 3129-3137.

Xi. Probe Method (Line Probe assay (LiPA));
Cooksey, RC, GP Morlock, S. Glickman, and JT Crawford. 1997. Evaluation of a line probe assay kit for characterization of rpoB mutations in rifampin-resistant Mycobacterium tuberculosis isolates from New York City. J. Clin. Microbiol. 35: 1281-1283.

 De Beenhouwer, H., Z. Lhiang, W. Jannes, L. Nfijs, L. Machtelinckx, R. Rossau, H. Traore, and F. Portaels. 1995. Rapid detection of rifampin resistance in sputum and biopsy specimens from tuberculosis patients by PCR and line probe assay. Tubercle Lung Dis. 76: 425-430.

 XII. Method using phages (PhaB assay).

 Wilson, S. M., Z. Al-Suwaidi, R. McNerney, J. Porter, and F. Drobniewski. 1997. Evaluation of a new rapid bacteriophage-based method for the drug susceptibility testing of Mycobacterium tuberculosis. Nat. Med. 3: 465-468.

Chain extension methods and allele-specific PCR (I, II) require independent reactions to be performed according to the number of mutations studied (i.e., more than 30 tubes in the case of the rpoB gene) and, accordingly, a large number of the studied sample;
The methods for studying polymorphism (III, IV), PCR heteroduplex analysis (VIII), and the imperfect duplex method with RNA (IX) are laborious, take a lot of time, give an indirect conclusion about the type of mutation and require standard standards (for each mutation); in addition, for the imperfect duplex method, increased requirements are imposed on the absence of RNAase and it is laborious and unacceptable for the detection of GU duplex [12, 18];
Hybridization on microarrays (V) requires a complex computer calculation of the results and expensive consumables (microarray itself);
Direct sequencing (VI) requires isolation of pure culture, has a high cost and requires a sequencing device;
Structurally specific cleavage (X) and dideoxyfingerprinting (VII) methods require preliminary standardization (selection of conditions), which increases the complexity and the presence of standard standards. In addition, dideoxy fingerprinting is performed with a radioactive label.

 The probe method (XI) has a high cost and works on a limited number of mutations [18].

 The method using phages (XII) takes a large amount of time, because it is associated with the replication of phages and the subsequent registration of lysis on the culture of M.smegmatis, and is laborious.

 These disadvantages are eliminated in the present invention.

Advantages of the biochip hybridization method
The hybridization method on biochips compares favorably with the above methods with the ability to analyze directly from a clinical sample (in the case of pulmonary tuberculosis), the ability to determine several mutations present at the same time, low cost, short time to obtain the result, and does not require expensive equipment and highly qualified personnel. In addition, data obtained using the hybridization method can be used for epidemiological genotyping.

SUMMARY OF THE INVENTION
A method has been developed for the accelerated determination of nucleotide substitutions in the DNA of tuberculosis mycobacteria, leading to the resistance of the pathogen to rifampicin. The method is based on the amplification of a rpoB fragment of a gene by two-stage PCR to obtain a single-stranded fluorescently labeled product and hybridization of the amplified labeled product on a biochip with subsequent registration and interpretation of hybridization results. The method involves the use of a specific asymmetric PCR pair of primers at the asymmetric PCR stage, one of which is fluorescently labeled. The use of this pair of primers allows amplification of a single-stranded fluorescently labeled fragment of a small size, which is able to penetrate into the pores of the gel and enter into hybridization interaction. The size of the amplified single-stranded fluorescently labeled fragment using the proposed pair of primers is about 127 bp Amplification of the rpoB gene fragment is carried out using directly the material of a clinical sample, which may include sputum, exudate, flushing, or broncho-alveolar lavage. The hybridization step is carried out using a biochip containing a set of differentiating oligonucleotides, which allows the detection of point mutations in the rpoB gene region with high specificity. The arrangement of oligonucleotides provides the ability to visually assess the presence of a mutation and its exact localization. The developed hybridization and washing conditions provide a high degree of differentiation between perfect and imperfect hybridization duplexes and allow the procedure to be carried out in an ordinary diagnostic laboratory. The registration of hybridization results can be carried out both visually and using a photographing device. The interpretation of the recorded results is performed by comparing the fluorescence intensity of the signals obtained with perfect and imperfect hybridization. The method can be applied for epidemiological genotyping.

 Based on the method for determining nucleotide substitutions in DNA, a method for diagnosing rifampicin resistance of tuberculosis mycobacteria is proposed.

 To implement the two indicated methods, a special differentiating biochip has been developed, which allows one to determine nucleotide substitutions in the DNA of tuberculosis mycobacteria, leading to the resistance of the pathogen to rifampicin. The biochip is a microarray with cells in which, in accordance with table. 1 and 2, a set of oligonucleotides was immobilized, which makes it possible to detect point mutations in the rpoB gene region with high specificity. Oligonucleotides are immobilized in such a way that the top row of the biochip includes oligonucleotides complementary to wild-type DNA sequences, and the corresponding column below each cell includes oligonucleotides complementary to mutant-type DNA sequences responsible for replacing one amino acid. The scheme of immobilization of oligonucleotides allows you to visually assess the presence and localization of a mutation.

List of figures
FIG. 1. The location of oligonucleotides on a differentiating biochip. The number enclosed in the square of the gel cell corresponds to the oligonucleotide number in the table. 2.

 FIG. 2. The arrangement of oligonucleotides that detect amino acid substitutions on the biochip. In the upper row are amino acids corresponding to the “wild” (non-mutated) type of DNA.

 FIG. 3. The arrangement of oligonucleotides that detect point nucleotide substitutions on the biochip, leading to the replacement of the amino acid residue. The sequence number corresponds to the first nucleotide from the triplet. Del-deletion of 6 nucleotides at positions 1294-1299.

 FIG. 4. Hybridization pattern of a DNA sample isolated from a rifampicin-sensitive strain of Mycobacterium tuberculosis, recorded on a fluorescence microscope.

 FIG. 5. Hybridization pattern of a DNA sample isolated from a rifampicin-resistant strain of Mycobacterium tuberculosis, recorded on a fluorescence microscope.

 FIG. 6. Hybridization pattern of a DNA sample isolated from a rifampicin-resistant strain of Mycobacterium tuberculosis, recorded on a fluorescence microscope.

Disclosure of the invention
The objective of the present invention is the creation of an accelerated method for the detection of point nucleotide substitutions in the DNA of Mycobacterium tuberculosis, leading to the emergence of resistance of the pathogen to rifampicin on biochips.

 The claimed method suggests the use of: obtaining a single-stranded fluorescently labeled fragment of the rpoB gene, mutations in which lead to the emergence of resistance of tuberculosis mycobacteria to rifampicin, directly from a clinical sample (sputum in case of pulmonary tuberculosis); hybridization of the obtained fragment to the oligonucleotide biochip; registration on a photographing device.

Schematic diagram of the detection of rifampicin-resistant strains of mycobacterium tuberculosis on a biochip
A clinical sample (in the case of a pulmonary form - sputum) is diluted with alkali and N-acetyl-L-cysteine, boiled with a detergent to provide access to DNA and decontamination (disinfection) of the sample. A specific fragment of the rpoB gene is accumulated by PCR, then a single chain and labeled (suitable for hybridization) asymmetric PCR product is obtained using a fluorescently labeled primer.

 The resulting product is hybridized to a differentiating biochip containing oligonucleotides that are complementary to gene sequences with or without mutations (wild type).

 The studied DNA fragments form perfect hybridization duplexes with the corresponding oligonucleotides. With all other oligonucleotides, the studied DNA fragments give an imperfect duplex. The differentiation of perfect and imperfect duplexes is carried out by comparing the fluorescence intensities of these duplexes. The signal intensity of a perfect duplex is higher than that of an imperfect one. (Comparing the above values of signal intensities, determine which duplex was formed: perfect or imperfect). Using the arrangement of oligonucleotides on the biochip, mutations inherent in the studied DNA sequence are determined, and a conclusion is made on the resistance (or sensitivity) of the studied object to rifampicin.

The implementation of the method of the invention
Clinical Sample Processing
A clinical sample (sputum, exudate, flush, broncho-alveolar lavage) 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 40 minutes. A pH of 6.8 phosphate buffer was added to the sample at a ratio of 1: 5 by volume and centrifuged for 30 min at 3000 rpm. When using cerebrospinal fluid, it was centrifuged in an Eppendorf centrifuge for 10 min at 10,000 rpm. During blood analysis, the lymphocyte fraction was isolated according to the generally accepted method using ficoll. Further processing of the samples was carried out identically.

 Cell pellet was suspended in 1.5 ml TE buffer, pH 8.0, and precipitated at 3000 rpm for 30 minutes. The washing was repeated once more.

To the resulting precipitate was added 30-70 μl of TE buffer, pH 8.0, containing 1% (v / v) Triton X-100, and kept in a dry thermostat at 95 ^° C for 15 minutes.

 The sample was centrifuged at 10,000 rpm for 10 min in an Eppendorf centrifuge, and the supernatant (3 μl) was used for PCR.

 Amplification of a fragment of the rpoB gene and obtaining a single-chain labeled product by PCR.

 In 50 μl of the PCR mixture add 3 μl of the sample obtained in paragraph 5.

The composition of the PCR mixture
IX PCR buffer: 50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25 ^o C), 0.1% Triton ^® X-100 (Promega);
1.5 mM MgCl ₂ (Promega)
200 μM each dNTP (Sigma)
5 pmol of each primer (rpo105 and rpo273)
1.5 units Thermostable Taq DNA Polymerase (Promega)
Primers
The rpo105 primers (5'-cgt gga ggc gat cac ace gca gac gtt g) and rpo273 (5'-gac etc cag ccc ggc acg etc acg t) flank the 193 bp rpoB gene fragment. (positions 1224 - 1416, Ac. N L27989), they were synthesized using the standard phosphoamidite procedure and purified by reverse phase HPLC.

1. Amplification is carried out on a MiniCycler programmable thermostat (MJ Research, USA) with the following mode: 95 ^o C - 30 s, 68 ^o C - 40 s, 72 ^o C - 20 s; 33 cycles (in the first cycle, the time of denaturation was increased to 5 minutes, in the last time of elongation to 5 minutes).

 2. 1 μl (~ 100 ng) of the amplified double-stranded DNA preparation was transferred to 100 μl of a PCR mixture to obtain a single-stranded labeled fragment of the rpoB gene.

The composition of the PCR mixture
IX PCR buffer: 50 mM KCl, 10 mM Tris-HCI (pH 9.0 at 25 ^o C), 0.1% Triton ^® X-100 (Promega);
1.5 mM MgCl ₂ (Promega)
200 μM each dNTP (Sigma)
10 pmol primer 1272f
1 pmol of primer 1378r
1.5 units Thermostable Taq DNA Polymerase (Promega)
Primers
Primer 1272f (5'-cgc cgc gat caa gga gtt gt ct) contained at the 5'end of the C ₆ aminomodifier (Glen Research Corp., VA), to whose amino group Texas Red ^® -sulfonyl chloride (Molecular Probes Inc., Eugene , OR) according to the protocol recommended by the manufacturer.

 Primer 1378r (5'-tca cgt gac aga ccg ccg gg) was synthesized using a standard phosphoamidite procedure. Both primers were purified by reverse phase HPLC.

Primers flank a 127 bp rpoB gene fragment. (Items 1272 - 1398 Ac. N L27989)
3. Amplification is carried out as described in paragraph 6, with the exception of: instead of 30 cycles, 36 are carried out; the annealing temperature instead of 68 - 65 ^o C. 12 μl of the obtained product (a mixture of double-stranded, single-stranded DNA and primers) is used for hybridization on a biochip
Hybridization of the amplified labeled product on a biochip
4. 12 μl of the sample obtained in asymmetric PCR (~ 1.2 μg ssDNA) is used for hybridization on a biochip in a buffer of the following composition: 1M GuSCN; 50mM HEPES, pH 7.5; 5mM EDTA, pH 7.0. Hybridization is carried out in a commercially available hybridization chamber with a volume of 30 μl (Sigma) overnight (16-18 hours) at 37 ^° C. Washing is carried out three times in buffer: 6.7x SSPE; 10% Tween 20 at 37 ^o C.

Registration and interpretation of hybridization results
5. Fixation of the hybridization picture is carried out using a photographing device. For research (non-practical) work, the measurement of the fluorescence signal and the fixation of the hybridization pattern are carried out on an automatic complex consisting of a fluorescence microscope, 512x512 CCD camera (3x3 mm ² field of view), thermal controller and computer. WinView (Princetone Instruments, USA) is used for scanning and subsequent image processing; fluorescence signal intensity is processed using programs using the LabVIEW interface (National Instruments, Austin, Texas).

 6. The interpretation of the results is as follows. Inside each column of gel cells, the intensities of the fluorescent signals are visually (or using software). If the signal intensity of the upper cell is greater than in the cells located below it, then according to this column (amino acid residue), the studied sample is referred to the wild type, i.e. not mutated. If one of the downstream cells (inside one column) has a higher intensity than the topmost cell in the column, then the studied nucleic acid sample has a point mutation. (The type and location of the mutation is established by comparison with the arrangement of oligonucleotides on the biochip). If, for each column (amino acid residue), the studied sample is defined as having no mutations, the studied sample is classified as wild-type, and it is concluded that tuberculosis insensitive to rifampicin was not detected in the studied clinical sample of mycobacteria. If at least one column the studied sample is defined as having a point nucleotide mutation, the studied sample is referred to the “mutant type”, and it is concluded that tuberculosis mycobacteria that are sensitive (resistant) to rifampicin are found in the studied clinical sample.

 Oligonucleotide biochip for the detection of rifampicin-resistant strains of mycobacterium tuberculosis.

 Oligonucleotides for immobilization on a biochip were synthesized on a 394 DNA / RNA synthesizer (Applied Biosystem) using a standard phosphoamidite procedure. The 3 ′ end of the oligonucleoids contained a CPG spacer (Glen Research, VA).

 The biochip was manufactured as previously described [Proc. Natl. Acad Sci. USA 93, 4913-4918]. A microarray containing cells measuring 100x100x20 μm was prepared by photopolymerization of a 5% polyacrylamide gel, as described previously [ibid.].

 The gel was activated with 2% trifluoroacetic acid at room temperature for 10 minutes, washed with water and dried. Next, the gel was sequentially processed in Repel-Silane (2% solution (weight / volume) of dimethyldichrolosilane in 1,1,1-trichloroethane, LKB-Produkter AB, Bromma, Sweden) for 30 s; dichloromethane - 10 s; ethanol (95 vol.%) - 10 s; washed with water for 3 min and dried.

A 1 mM oligonucleotide solution was applied twice with a robot needle in an amount of 1 nl (final concentration 2 μM). To stabilize the covalent bonds between the 3'-terminal amino groups of the oligonucleotides and the aldehyde groups of the gel, the matrix was placed in a 0.1 M solution of the pyridine-borane complex (Aldrich Chemical Co., Inc., Milwaukee, WI) in chloroform, the upper aqueous phase was layered and held 12-16-16 hours at room temperature. Next, the biochip was washed twice with ethanol (95 vol.%) And water. Unreacted aldehyde groups were reduced with a freshly prepared solution of 0.1 M NaBH ₄ (Aldrich) for 20 minutes at room temperature. The biochip was washed with water, dried and stored at room temperature.

Biochip structure
The biochip contains 31 immobilized oligonucleotides, a list of which is presented in table. 1 and 2. The upper row of the biochip includes oligonucleotides complementary to wild-type DNA sequences (from rifampicin-sensitive mycobacterium tuberculosis). Under each cell complementary to wild-type DNA, there are cells immobilized by oligonucleotides complementary to mutant-type DNA sequences (from rifampicin-resistant mycobacterium tuberculosis). Each column of cells contains a set of oligonucleotides responsible for replacing one amino acid (the number of which is shown above the upper row of cells in Fig. 2). The arrangement of oligonucleotides on the biochip is shown in FIG. 1. The biochip is capable of detecting 7 amino acid substitutions (see Fig. 2) or 22 corresponding nucleotide substitutions (see Fig. 3); one deletion containing 2 amino acids (corresponding to 6 nucleotides).

Examples
Example 1
Determination in a clinical sample of mycobacterium tuberculosis sensitive to rifampicin (wild-type DNA) by biochip hybridization
In FIG. Figure 4 shows the hybridization pattern of a DNA sample isolated from a rifampicin-sensitive strain of Mycobacterium tuberculosis, recorded on a fluorescence microscope.

 Inside each column of cells, the upper cell has a greater fluorescence signal intensity than the cells located below. Therefore, the DNA of the object under study forms perfect hybridization duplexes with oligonucleotides complementary to the wild-type DNA sequence (a strain of tuberculosis mycobacterium sensitive to rifampicin). It follows that in the studied clinical sample of mycobacteria resistant to rifampicin, it was not found.

Example 2
Determination of rifampicin-resistant tuberculosis mycobacteria (a DNA having a mutation) in a clinical sample by biochip hybridization
In FIG. Figure 5 shows the hybridization pattern of a DNA sample isolated from a rifampicin-resistant strain of Mycobacterium tuberculosis, recorded on a fluorescence microscope.

 Inside each column of cells, the upper cell has a greater fluorescence signal intensity than the cells located below. The exception is the formation of a perfect duplex in the cell indicated by the arrow. The signal intensity in this cell exceeds the intensity recorded in the upper cell of this column. Therefore, the DNA of the studied object has a point nucleotide substitution A> T at position N 1322, which leads to the replacement of the amino acid Asp by Val at position N 516 and leads to the resistance of the studied strain to rifampicin. It is concluded that rifampicin-resistant mycobacterium tuberculosis was found in the studied clinical sample. Resistance is due to the replacement of the amino acid Asp by Val at position N 516.

Example 3
Determination of rifampicin-resistant tuberculosis mycobacteria (a DNA having a mutation) in a clinical sample by biochip hybridization
In FIG. Figure 6 shows the hybridization pattern of a DNA sample isolated from a rifampicin-resistant strain of Mycobacterium tuberculosis, recorded on a fluorescence microscope.

 Inside each column of cells, the upper cell has a greater fluorescence signal intensity than the cells located below. The exception is the formation of a perfect duplex in the cell indicated by the arrow. The signal intensity in this cell exceeds the intensity recorded in the upper cell of this column. Therefore, the DNA of the studied object has two point nucleotide substitutions C> T at position N 1351 and A> G at position N 1352, which leads to the replacement of His amino acid by Cys at position N 526 and leads to the resistance of the studied strain to rifampicin. It is concluded that rifampicin-resistant mycobacterium tuberculosis was found in the studied clinical sample. Resistance is due to the replacement of His amino acid by Cys at position N 526.

 The above examples are preferred, are intended only to confirm the feasibility of the invention and should not be the basis for limiting the scope of the claims of the Applicant. A person skilled in the art will easily find the possibilities of other embodiments of the invention, which certainly fall within the scope of the Applicant’s claims, as set forth in the claims below.

 The presented invention allows to quickly determine rifampicin-resistant forms of mycobacterium tuberculosis, including directly using the material of a clinical sample. The method compares favorably with existing analogues by its simplicity of execution and low cost. The use of a biochip containing a set of differentiating oligonucleotides also allows typing (marking) of tuberculosis mycobacteria at the genotypic level.

Claims (11)

 1. A method for accelerated determination of nucleotide substitutions in the DNA of mycobacterium tuberculosis, leading to the resistance of the pathogen to rifampicin, including: (a) amplification of the rpoB gene fragment and obtaining a single-chain fluorescently labeled product by PCR; (b) - preparation of a biochip for the determination of rifampicin - resistant mycobacteria; (c) - hybridization of the amplified labeled product on a biochip; (d) - registration; and (e) - interpretation of the results of hybridization by comparing the intensity of fluorescent signals obtained with perfect and imperfect hybridization.  2. The method according to claim 1, characterized in that in step (a) a specific primer pair is used, one of which is fluorescently labeled, the use of which pair of primers allows amplification of a single-stranded fluorescently labeled fragment of a small size that can penetrate into the pores gel and enter into hybridization interaction.  3. The method according to claim 2, characterized in that the size of the amplified single-stranded fluorescently labeled fragment is about 127 bp  4. The method according to claim 1, characterized in that the amplification of the rpoB gene fragment is carried out using directly the material of a clinical sample.  5. The method according to claim 4, characterized in that the clinical sample includes sputum, exudate, flush or broncho-alveolar lavage.  6. The method according to claim 1, characterized in that at stage (b) a biochip is used containing a set of oligonucleotides, the use of which allows the detection of point mutations in the rpoB region of the gene with high specificity, and the order of placement of oligonucleotides provides the ability to visually assess the presence of a mutation and its exact localization.  7. The method according to claim 1, characterized in that at stage (c) the hybridization and washing conditions are used, which provide a high degree of differentiation between perfect and imperfect hybridization duplexes, which allows the procedure to be carried out in an ordinary diagnostic laboratory.  8. The method according to claim 1, characterized in that the registration of the results at stage (g) is carried out visually.  9. The method according to claim 1, characterized in that the registration of the results in stage (g) is carried out using a photographing device.  10. A method for diagnosing the resistance of mycobacterium tuberculosis to rifampicin by determining nucleotide substitutions in the DNA of the pathogen, leading to the emergence of resistance, according to claim 1.  11. The biochip for determining nucleotide substitutions in the DNA of mycobacterium tuberculosis, leading to the resistance of the pathogen to rifampicin, according to claim 1, which is a microarray with cells in which a set of oligonucleotides with the sequences shown in table. 1 and 2, for detecting point mutations in the rpoB region of the gene, the upper row of the biochip containing oligonucleotides complementary to wild-type DNA sequences, and the corresponding column under each cell contains oligonucleotides complementary to mutant-type DNA sequences responsible for replacing one amino acid.

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