RU2163638C1 - Method of detection of dna of tuberculosis mycobacterium complex with differential revealing mycobacterium tuberculosis dna and reagent set for its realization - Google Patents

Abstract

FIELD: molecular biology, genetic engineering, medicine (phthisiology), veterinary science. SUBSTANCE: invention proposes method and set based on a single-round multiprimer polycyclic amplification by polymerase chain reaction method in total reaction mixture. The latter has, in part, glycerol, two targets of genome DNA and nucleotide sequence as an internal standard. This ensures to estimate effectiveness of polymerase chain reaction on DNA of the sample to be analyzed and eliminate false-negative data of analysis. One of taken target is fragment of RD1 genomes that is typical for all species of tuberculosis mycobacterium complex (with exception for M. bovis BCG) involving gene site that encodes protein ESAT-6; the second target is fragment of gene mtp 40 showing specificity for M. tuberculosis. Proposed set has: (1) concentrated reaction mixture; (2) thermostable DNA-polymerase (5 U/mcl); (3) ionized sterile water; (4) mineral oil; (5) (+)-control DNA (M. tuberculosis DNA). Invention ensures to carry out differential detection of mycobacteria of species M. tuberculosis among other species of tuberculosis mycobacterium complex and reveal the above indicated mycobacteria in biological specimens obtained from humans, agriculture and wild animals, estimate effectiveness of therapy and disinfection measure. Invention is designated for detection of tuberculosis mycobacterium complex where species M. tuberculosis, M. bovis, M. microti and M. africanum belong. EFFECT: improved method of detection. 10 cl, 2 tbl, 1 ex

Description

 The invention relates to molecular biology, genetic engineering, medicine (phthisiology), veterinary medicine and is intended for the detection of Mycobacterium tuberculosis complex (MTK), which includes M. tuberculosis, M. bovis, M. microti and M.africanum. The invention allows for the differential identification of mycobacteria belonging to the species M. tuberculosis, among other types of MTK. In Russia and the CIS countries, where two types of MTK species, pathogenic for humans, are predominantly M. tuberculosis and M. bovis, the use of the invention allows for the differential identification of human and bovine mycobacteria.

 The invention can be used to identify the above mycobacteria in biological samples obtained from humans, agricultural and wild animals.

 The invention allows to detect mycobacteria in clinical samples (sputum, urine, scraping of epithelial cells, cerebrospinal fluid, rinsing water of the bronchi and stomach, blood, biopsy material), as well as in other samples (swabs from instruments, wall surfaces, floor of premises; in meat and dairy products, etc.).

 The invention can be used to evaluate the effectiveness of therapy and disinfection measures.

 The invention can be used in scientific research, for example, when studying the drug resistance of mycobacteria, determining the growth qualities of solid and liquid media, to determine the bactericidal effect on mycobacteria of various physical and chemical factors, etc.

 The well-known "Method of indicating pathogens of tuberculosis M. tuberculosis and M. bovis" (patent application N 94017389/13, IPC 6 C 12 Q 1/68, A 61 K 39/04, publ. 06/10/96). The essence of the method lies in the fact that the detection of pathogens of tuberculosis is carried out by sequential two-stage (two-raid) amplification of a portion of the gene encoding the 64-kilodalton protein M. bovis BCG, specific for the species M. tuberculosis and M. bovis, and two pairs are used in the polymerase chain reaction primers, one of which flanks the amplification site of the other pair of primers, and makes judgments about the presence of pathogens in the sample when bands characteristic of amplicons are detected on an agarose gel electrophoregram, synthesis using both pairs of primers.

Regarding this method, the following (disadvantages) can be noted:
1) firstly, during PCR sequentially, in two stages, the probability of contamination of the reaction mixture with amplicons obtained in the first stage of the reaction increases significantly;
2) secondly, PCR in two stages increases the duration of the analysis;
3) thirdly, since the gene encoding the 64-kilodalton protein is also characteristic of a number of vaccine strains of M. bovis BCG, the method based on the detection of this gene does not allow differentiating people and animals with tuberculosis from individuals recently vaccinated or revaccinated M. bovis BCG;
4) fourthly, the method does not allow to differentiate two types of tuberculosis pathogens M.tuberculosis and M.bovis, mainly prevalent in Russia and the CIS countries, which seems important both for rational treatment of patients and for assessing and predicting the epidemiological situation;
5) fifthly, since the method does not provide for the use of an internal standard for PCR, it is not possible to track false negative results of the analysis due to the presence of thermostable DNA polymerase inhibitors in some of the analyzed DNA samples;
6) sixth, the method does not allow a relative quantitative assessment of the content of tuberculosis pathogens in the analyzed sample;
The method described in the article "Differential detection of tuberculosis pathogens in clinical material using a two-stage polymerase chain reaction" by A. B. Beklemishev and E. M. was adopted as a prototype of the method. Good (Bulletin SB RAMS, 1998, N 3, p. 76-85).

 The article describes a method for detecting tuberculosis pathogens (MTK) with differential detection of M. tuberculosis, which, like the proposed method, allows differential detection of M. tuberculosis and M. bovis in the Russian Federation and the CIS. The method is based on the use of two-stage PCR with simultaneous (in one reaction mixture) amplification of two targets. One of them is a reliable specific genetic marker of the species M. tuberculosis - this is the mtp40 gene sequence. Another is a fragment of the sequence of the insertion element IS6110 specific for MTK.

 The disadvantages of the method are similar to those described in paragraphs. 1,2,3,5,6 of the first analogue.

For the prototype of the kit, the Politub kit was adopted, which is a set of reagents for detecting the DNA of mycobacterium tuberculosis complex in biological samples by PCR (TU 9398-410-17253567-97). The kit includes:
1) a reaction mixture containing a solution of dNTP, a solution of specific primers, PCR buffer, dye (cresol red);
2) thermostable DNA polymerase (Taq polymerase);
3) deionized water;
4) mineral oil;
5) DNA control (M. tuberculosis DNA).

 A method for detecting MTK using the Politub kit is also based on amplification of a portion of the MPB 64 protein gene.

 The disadvantages of this kit and the method for detecting MTK DNA are similar to those described for the first analogue.

1. SUMMARY OF THE INVENTIONS
The task of the claimed invention is to provide a highly specific, sensitive, but simpler and faster method and an appropriate set of reagents for the detection of genomic DNA of representatives of four types of MTK (M. tuberculosis, M. bovis, M.microti, M.africanum) with differential identification of M. tuberculosis that make it possible not to detect the vaccine strain M. bovis BCG and allow visual semi-quantitative assessment of the number of causative agents of tuberculosis in the analyzed material.

 The proposed method and kit are based on single-round, multi-primer, multi-cycle amplification by PCR in a common reaction mixture, which contains, in particular, glycerin, two sections (targets) of genomic DNA and a nucleotide sequence that serves as an internal standard and allows judging about the effectiveness of PCR for DNA analyzed sample and isolate false negative results of the analysis. A fragment (the RDI regions of the MTK genomes, including the portion of the ESAT-6 protein gene (Harboe M., Oettinger T., Wiker HG, Rosenkrands I., and Andersen P. Evidence for occurrence of the ESAT-6 protein in Mycobacterium tuberculosis and virulent Mycobacterium bovis and for its absence in Mycobacterium bovis BCG // Infect, lmmun / -1996. V.64.- P. 16-22), as the second is a specific fragment of the mtp 40 gene specific for M. tuberculosis (Portillo PD, Murillo LA, Patarroyo MEA; amplification of species-specific DNA fragment of Mycobacterium tuberculosis and its possible use in diagnosis // J. Clin. Microbiol. -1991 / -V .29. -P.2163-2168).

The proposed kit contains: 1) a concentrated reaction mixture ("cattle" -concentrated reaction mixture), which includes: 0.10 mM each of 4 dNTPs (dATP, dGTP, dCTP, dTTP), 33.2 mM (NH ₄ ) ₂ SO ₄ , 134 mM Tris-HCl (pH 8.8), 3.0 mM MgCl ₂ , 0.02% Tween 20, 20% (v / v) glycerol, 0.4 μM each of P90 primers , P92, P93, and P94 matched to the target nucleotide sequences of the MTK genomes, i.e. to the fragment of the RDI region of MTK genomes, including the ESAT-6 protein gene region and to the mtp-40 gene fragment, as well as BC DNA (N 1 or N 2 or N 3) at a concentration of 5 · 10 ² -2.5 · 10 ³ copies / ml cattle and cresol red dye (1 mg / ml cattle);
2) thermostable DNA polymerase (5 units / μl);
3) deionized, sterile water;
4) mineral oil;
5) (+) - control-control DNA (M. tuberculosis DNA).

 - the quantities of which are specified in clause 2.2.

1. 1. Characterization of targets selected for amplification
In this invention, two nucleotide sequences of MTK genomes are selected as targets for amplification in a PCR system. Both targets are used for simultaneous amplification in a common reaction mixture. The first target (target M) is a fragment of the nucleotide sequence of the MTP 40 protein gene, which is species-specific for M. tuberculosis and is present in the vast majority of its isolates, as well as in some isolates of the M.africanum species, not common in Russia and the CIS countries. The second target (target E) is a fragment of the nucleotide sequence of the RDI MTK genomes, including a portion of the ESAT-6 protein gene. This protein is specific for all types of MTK, with the exception of M. bovis BCG. Such selection of targets makes it possible to judge by the results of PCR in each positive sample whether mycobacteria belong to M. tuberculosis or not belong to this species, i.e. on belonging to other types of MTK. Since two types of tuberculosis pathogens, M. tuberculosis and M. bovis, are mainly distributed in Russia and the CIS countries, the use of the proposed method and kit allows us to differentiate these species among themselves, i.e. identify both of these species, which may be useful for assessing the epidemiological situation of tuberculosis. The fact that this selection of targets excludes the possibility of determining the vaccine strain of M. bovis BCG is also an additional advantage, especially given the vaccination of the majority of the population.

1.2. Primer structures used to amplify M and E targets and DNA of various internal standard models
The selection of primers was carried out using the program "Oligo".

Primers were selected (designed) in such a way that:
1) the temperatures of their annealing (hybridization) with the corresponding nucleotide sequences of the targets and DNA of the internal standard were the same or close, because PCR according to the proposed method is carried out in one stage in one reaction mixture (and in one container),
2) all three amplicons differed in size, and therefore in electrophoretic mobility, and, thus, were clearly discriminated after gel electrophoresis.

 The structures of some of the possible primers used in the proposed method and kit are shown in Table 1. The primers for target E, as can be seen from the table, are also used for amplification of DNA of the internal standard of model N 1 (cloned nucleotide sequence) and model N 3. K model N 2 of the internal standard, a separate pair of primers was selected.

1.3. Development and production of DNA of the internal standard (BC)
As already mentioned, in approximately 10% of the samples, when analyzing them for the presence of MTK DNA, PCR inhibitors are detected. In the proposed method and test system to solve the problem of tracking false-negative results, the DNA of the internal standard, designed for co-amplification with the DNA of the analyzed clinical sample, is introduced into the reaction mixture.

 The internal standard (BC) used in PCR is a specific DNA preparation containing a fragment amplified by the PCR process whose amplicon size differs from the sizes of amplicons synthesized on the target nucleotide sequences of the detected genome. BC DNA is introduced into the composition of the reaction mixture and, thus, BC present in both experimental and control samples is amplified in them during PCR analysis together with the targets of the analyzed DNA or control DNA. If the preparation of the analyzed DNA contains impurities that inhibit the activity of thermostable DNA polymerase, this, in turn, leads to complete or partial suppression of amplification of both the DNA of the test sample and the DNA of BC. The results of the analysis of samples in which there is no amplification of both the studied DNA and DNA of the BC should be considered as false negative.

 As the BC DNA, one of the target nucleotide sequences can be used, in which the internal part of the nucleotide chain has been delegated by genetic engineering, or, on the contrary, a fragment of any DNA is inserted. The target modified in this way can be used directly as a BC, or previously cloned as part of a plasmid vector, and, in this case, a recombinant plasmid is used as a BC. Amplification of the modified target of the BC is carried out using the same pair of primers that is used to amplify the original target in the genome of the detected pathogen. In order to avoid competition between the analyzed DNA and the DNA of the BC for the same primers, which can affect the results of the analysis, as well as to avoid the formation of hybrid DNA between the chains of amplicons of the BC and the target, heterologous DNA can be used as a BC, but with the corresponding pair primers (BC model N 2) or a pair of primers used to amplify one of the targets (BC model N 3).

 Model N 1 of the BC is designed in such a way that the BC as one of the DNA targets (target E) is amplified in the presence of primers p93 and p94. In this case, an amplicon of 231 bp is produced. (see table 1).

Getting aircraft model N 1 is carried out according to the following scheme:
1. In the first step, a fragment of the RDI nucleotide sequence of the M. tuberculosis genome is amplified, the primary structure A of which (positions 1914 to 2554, the nucleotide residue of the RDI region) is presented at the end of the description. The fragments to which primers p80 and p81 are selected (see Table 2) are underlined. The size of the synthesized amplicon is 641 bp Sequences in italics indicate primers N93 and N94 (see Table 1) used both for amplification of the RDI fragment for detection of mycobacteria DNA in this test system (the size of the synthesized amplicon is 545 bp) and for the amplification of BC DNA N 1 and N 3. SacI, PstI, and TaqI restriction enzyme recognition sites are shown in bold.

 2. In the second step, the amplicon is digested first with restriction enzymes SacI and PstI, and then with restriction enzyme TaqI. Restrictions are separated by electrophoresis in 5% PAG and the 5 'and 3' terminal fragments of the amplicon are eluted from the gel and ligated using T4 phage DNA ligase.

 3. At the third stage, the mixture of ligated fragments is introduced as template DNA into the PCR system containing primers p80 and p81. Amplicon 327 bp purified by electrophoresis in 5% SDS page followed by elution. The primary structure B of this amplicon is shown at the end of the description.

4. At the final stage, the connectors (dC) _15-20 are added to the 3'-ends of the amplicon using terminal deoxynucleotidyl transferase and cloned in the plasmid vector pBluescript II SK ⁽⁺⁾ in E. coli cells. The vector is pre-hydrolyzed with restriction enzyme PstI and then the connectors (dG) _{15-30 are} extended to the 3'-ends.

 A recombinant plasmid containing an amplicon of 327 bp in size was diluted to a concentration of 10-100 copies in 5 μl and used in PCR as model N 1 BC. Amplicon 231 p in size was synthesized by amplification of BC 1 DNA with primers p93 and p94. n

DNA DNA of model N 2 is obtained as follows:
1. At the first stage, a fragment of the nonstructural region (NS) of the human hepatitis C virus RNA genome (positions 43 to 339 of the nucleotide residue of the genome) is subjected to reverse transcription using M-MLV revertase in the presence of p60 and p61 primers (see table 2) .

 2. At the second stage, amplification of the cDNA synthesized at the first stage is carried out in a PCR system in the presence of p60 and p61 primers. Synthesized amplicon measuring 297 bp after appropriate dilution with a TE buffer to a final concentration (10-100 molecules in 5 μl of a solution), the model N 2 is used in the test system. The DNA structure of N 2 is presented at the end of the description (sections of the genome fragment to which p60 primers are selected and p61 are underlined).

BC DNA of model N 3 is obtained based on BC DNA N 2 as follows:
1. At the first stage, BC N 2 DNA is amplified in a PCR system in the presence of p116 and p117 primers (see Table 2) to obtain an amplicon of 324 bp in size. (pvs3).

 2. At the second stage, amplification of the pvc3 amplicon synthesized at the first stage is carried out in the presence of primers p93 and p94 (see table 2). Synthesized amplicon measuring 345 bp after appropriate dilution with a TE buffer to a final concentration (10-100 molecules in 5 μl of a solution), the model N 2 is used in the test system. The structure of DNA N 3 is presented at the end of the description (sections of the genome fragment to which p60 primers are selected and p61 are underlined, and areas corresponding to primers p93 and p94 are shown in bold).

 An amplicon, corresponding in size to the BC DNA used in the test system (N 1, N 2 or N 3), should be observed after PCR in all samples, including a sample containing a negative control. The absence of BC DNA amplification products is evidence of inhibition of PCR and, therefore, that a negative result may be false. Amplification of known VS dilutions of any model can be used to semi-quantify the number of tuberculosis pathogens in the analyzed material.

1.4. PCR conditions for the detection of MTK DNA and differential detection of M. tuberculosis DNA
The PCR conditions (temperature, time, number of cycles, the amount of glycerol and the internal standard) were optimized (mutually selected) so as to obtain a single-tube multi-primer multi-cycle amplification of both diagnostic targets (E and M) together with the DNA of one of the BC models and without non-specific amplification products. The use of 10% glycerol in the reaction mixture allowed us to reduce the temperature of DNA denaturation and annealing of primers by 4 degrees and thus extend the enzyme's “life”. This, in turn, allowed: a) to increase the sensitivity of the method by increasing the number of amplification cycles, and therefore the number of amplicons, without additional introduction of the enzyme into the reaction mixture, b) to carry out PCR in one stage, which is important to prevent cross-contamination of the analyzed samples accumulated amplicons.

 The amount of AC in the reaction mixture was selected so that it did not significantly affect the amplification of targets due to competition for dNTPs.

2. INFORMATION CONFIRMING THE POSSIBILITY OF CARRYING OUT THE INVENTION
The method is as follows:
The set can analyze the following types of biological material: sputum, urine, lavage fluid, pleural and cerebrospinal fluid, various exudates, blood. According to the processing method, clinical samples are divided into three groups: blood, liquid samples containing mucus, and other liquid samples.

2.1. Analysis
2.1.1. Preparation of samples for DNA isolation
A) Preparation of blood samples.

80 μl of peripheral blood from a finger is collected in a 1.5 ml plastic tube containing 30 μl of 3.8% sodium citrate, stirred and stored for no more than 3 days at + 4 ^o C. 900 μl of distilled water is added to a solution of citrated blood then 300 μl of 2M NaOH was stirred and incubated for 30 min at room temperature.

 B) Preparation of liquid samples containing mucus.

 If urine, lavage fluid, or some other sample contains mucus, it is diluted by adding a few drops of concentrated alkali NaOH and suspending it.

 About 1/4 of the 2M NaOH solution was added to the sputum sample and suspended until liquid. If after this the mucus remains, then it is diluted by adding a few drops of concentrated alkali NaOH and re-suspension.

 2.1.2. Isolation of DNA from samples.

 After the stages described in clause 2.1.1. all three groups of samples are treated equally. Samples are centrifuged at 3500gx20 min. and remove the supernatant. 1 ml of TE buffer (10 mM Tris-HCl, pH 8.0, 1mM EDTA) was added to the pellets, suspended and the material was transferred into 1.5 ml plastic tubes.

The pellet suspension in the TE buffer is centrifuged at 10000xg, the pellet is washed again with TE buffer 2 times and used to isolate DNA
1 ml of chloroform was added to the precipitate and stirred on a microtube shaker for 3 minutes. Then, 100 μl of TE buffer was added to the suspension with a micropipette and stirred again for 3 minutes. The resulting suspension is centrifuged at 8000-10000 rpm for 5 minutes, as a result of which it is divided into two phases, in the upper (aqueous) of which contains DNA.

2.1.3. PCR analysis
1. Make two drops of mineral oil (30 μl) from the tip per 1-200 μl into plastic tubes with a capacity of 0.5 ml.

2 Prepare the reaction mixture based on 1 sample: water - 10 μl, cattle - 15 μl, thermostable DNA polymerase - 0.4 μl. The resulting reaction mixture was thoroughly mixed. This reaction mixture has the following composition: 16.6 mM (NH ⁴ ) ₂ SO ₄ , 67 mM Tris-HCl (pH 8.8), 1.5 mM MgCl ₂ , 0.01% Tween 20, 0.05 mM each each of 4 dNTPs (dATP, dGTP, dCTP, dTTP), 0.2 μM each of the primers P90, P92, P93 and P94, 10% (v / v) glycerol, 10-100 copies of BC DNA and 2.5 units Taq or Tet-Z DNA polymerase.

 3. 5 μl of the DNA preparation isolated from the clinical sample is added to a separate tube under a layer of mineral oil.

4. Pipette into two separate tubes:
a) positive control - 3 μl of control DNA, (M. tuberculosis DNA), with a concentration of 5-200 M. tuberculosis genomes per 1 μl.

 b) negative control - 3 μl of deionized water.

 5. The tubes are centrifuged for 10,000 g x 5 sec.

6. The tubes are incubated at 100 ^o 3 minutes, and then placed in an ice bath.

 7. Pipette 25 μl of the reaction mixture into each tube of denatured DNA, including control tubes.

8. The tubes are centrifuged for 2-3 seconds at 5-10 thousand rpm and placed in an amplifier with the set "Pause" mode at +80 ^o C. PCR is carried out in the following temperature mode ^* :
94 ^o C, 2 min; 65 ^o C, 40 sec; 72 ^o C, 40 sec - 1st cycle; 94 ^o C, 40 sec; 65 ^o C, 40 sec; 72 ^o C, 40 sec - up to 7 cycles; 90 ^o C, 30 sec; 65 ^o C, 40 sec; 72 ^o C, 40 sec - up to 50 cycles; 90 ^o C, 30 sec; 65 ^o C, 2 min; 72 ^o C, 9 min - 51st cycle.

^* PCR is carried out on an MC-2 amplifier of the DNA-technology company (Moscow), in the "fast active regulation" mode.

2.1.4. Registration and interpretation of analysis results
After completion of the reaction, 10 μl of the reaction mixture was analyzed by electrophoresis in 5% PAG, or in a 1.5-2% agarose gel at a voltage of 10 V / cm for 1 hour, followed by staining of the DNA with ethidium bromide gel.

Interpretation of analysis results
Strip size 231 bp corresponds to amplification products of BC model N 1 artificially introduced DNA DNA into the reaction mixture. If BC model DNA N 2 or N 3 is used in the test system, bands corresponding to 297 bp DNA in their mobility will be detected on the gel. or 345 bp, respectively.

 Band size 402 bp corresponds to amplification products of a genetic marker characteristic only for M. tuberculosis species.

 545 bp band corresponds to the amplification products of a genetic marker that is strictly specific for all pathogenic mycobacteria of the tuberculosis complex (absent in the M.bovis BCG vaccine strain).

1. Negative control - only one band must be present on the gel, corresponding to an amplicon with BC DNA (231 bp for the BC model N 1, 297 bp for the BC model N 2, or 345 bp for the BC Model N 3)
2. Positive control - there should be three bands corresponding to amplicons (545 bp and 402 bp) from the genetic markers of the genomic DNA of mycobacteria and to the amplicon with the DNA of one of the BC models.

3. The analyzed samples:
a) the presence of only one band corresponding in size to the amplicon with the DNA of one of the BC models and the absence of 545 bp bands on the gel and 402 bp , indicates the absence of DNA of the causative agent of tuberculosis in the analyzed sample (negative result);
b) the presence of 544 bp bands on the gel and 401 bp, along with a strip corresponding to the size of an amplicon with the DNA of one of the BC models, indicates the presence of DNA of the pathogen M. tuberculosis in the clinical sample (positive result);
c) if only a strip of 544 bp in size is detected on the gel, along with a strip corresponding to the size of an amplicon with the DNA of one of the BC models, this means that the DNA of the pathogen M. bovis is present in the sample (positive result);
d) the absence of all bands on the gel, including the band corresponding to the size of the amplicon with the DNA of one of the BC models, indicates the presence of PCR inhibitors in the sample DNA preparation; this result should be interpreted as false negative (no reliable result).

 4. The appearance of the band 401 bp or 544 bp in the negative control, indicates cross-contamination of samples or contamination of kit components.

 2.2. An example of a specific kit.

Polymerase chain reaction kit (for 100 determinations)
1) The reaction mixture ("cattle") - 1500 μl
2) Thermostable DNA polymerase (5 units / μl) - 40 μl
3) bidistilled water - 1500 μl
4) Mineral oil - 3000 μl
5) DNA preparation of M. tuberculosis with a concentration of 200 genomes / μl as a positive control ("K +") - 50 μl
2.3. The results of the application of the proposed method and set.

 The proposed invention was applied to the analysis of clinical samples (sputum, blood, urine, gastric lavage) from 22 patients. Samples were obtained from the Central Clinical Hospital SB RAS.

 All samples were investigated in parallel by three methods: smear microscopy, cultivation on selective Levenshtein-Jensen medium, and using the proposed inventions.

 By the method of cultivation, the causative agent of tuberculosis was detected after 8-10 weeks in 5 samples (in 17 samples, it was not). In the same samples, DNA of the causative agent of tuberculosis was detected by PCR. Moreover, in one of them - M. bovis DNA (the method of selective media does not allow differentiating M. tuberculosis and M. bovis).

 By means of microscopy, the pathogen bacilli were detected only in three samples that yielded positive results by cultivation and PCR.

 Thus, the proposed method, carried out using the proposed kit, is not inferior in sensitivity to the cultivation method, but, in contrast to it, is a much faster method (8 hours instead of 8-10 weeks) and allows to differentiate the types of M. tuberculosis and M. bovis.

Claims (10)

1. A method for detecting DNA of Mycobacterium tuberculosis complex (MTK) M. tuberculosis, M. microti, M. africanum, M. bovis, with the exception of M. bovis BCG, with differential detection of M. tuberculosis DNA by amplification by polymerase chain reaction (PCR) two nucleotide sequences, one of which is specific for the genomes of all the above MTK representatives, and the other is for the M. tuberculosis genome, using a fragment of the MTP40 protein gene as a target for detecting M. tuberculosis DNA, characterized in that it is a target for detecting DNA all representative MTC is used region fragment RDI genome comprising the protein ESAT-6 gene portion, amplification is carried out by multipraymernoy multicycle PCR in a single step in the initial reaction mixture containing agent provides carry out the reaction at a temperature of not higher than 94 ^o C and a DNA internal standard, allowing identify cases of inhibition of thermostable DNA polymerase in each sample.  2. The method according to claim 1, characterized in that the agent providing PCR at a reduced temperature at the stages of DNA denaturation and annealing of the primers is glycerol with a concentration in the reaction mixture of 10%.  3. The method according to claim 1, characterized in that PCR is carried out in the presence of DNA of an internal standard, the amplification of which is carried out with primers to one of the determined targets or with own primers.  4. The method according to claim 1 or 3, characterized in that the DNA of the internal standard is introduced into the reaction mixture in an amount of 10 to 100 copies per 30 μl of the reaction mixture. 5. The method according to any one of the preceding paragraphs, characterized in that during amplification, the stage of DNA denaturation from 1 to 7 cycle is carried out at a temperature not exceeding 94 ^{o C.} , and from 8 to 51 cycle at a temperature not exceeding 90 ^o C.  6. A set of reagents for the detection of DNA of Mycobacterium tuberculosis complex (MTK) M. tuberculosis, M. microti, M. africanum, M. bovis, except for M. bovis BCG, with the simultaneous detection of M. tuberculosis DNA by multi-cycle single-stage multi-primer amplification by PCR, comprising a reaction mixture containing oligonucleotide primers to a target specific for the M. tuberculosis genome, for example, an mtp 40 gene fragment, for example, SEC ID 1 and SEC ID 2 primers in the sequence listing, and to a target specific for all types of MTK and for internal DNA standard (model 1 or 3), e.g. an example to a fragment of an RDI region containing a portion of an ESAT-6 protein gene, for example, SEC ID 3 and SEC ID 4 primers in a sequence listing; glycerin, DNA of the internal standard, which is used to evaluate the activity of thermostable DNA - polymerase in a mixture with the analyzed sample.  7. The kit according to claim 6, characterized in that it contains pairs of primers selected so that their annealing temperatures in the matrices are the same or close, and the sizes of their corresponding amplicons are varied so as to ensure a distinct separation on the electrophoregram.  8. The kit according to claim 6, characterized in that it contains the DNA of the internal standard (model 1) SEC ID 7 in the sequence list.  9. The kit according to claim 6, characterized in that it contains DNA of an internal standard (model 2) SEC ID 8 and primers for its amplification: SEC ID 5 and SEC ID 6 in the sequence listing.  10. The kit according to claim 6, characterized in that it contains the DNA of the internal standard (model 3) SEC ID 9 in the sequence list.

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