RU2409680C1 - Diagnostic technique for of mycobacterium tuberculosis strain sensitivity to aminoglycosides - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: diagnostic technique for Mycobacterium tuberculosis (MBT) strain sensitivity to aminoglycosides involves the stages of DNA recovery, polymerase chain reaction (PCR) based on DNA amplification of an analysed and sensitive MBT strains, PCR-product restriction, separation of restriction products in agarose gel and the following analysis of restriction profiles. MBT DNA is recovered from cultures produced from a fluid medium, heated for 20 min at 95°C in TE buffer with 1% triton pH 8.8. For conducting the PCR of the preset sequence, there is used a reaction buffer containing in 50 mcl: 10 mM of Tris-HCl pH 8.8; 50 mM of KC1; 0.5% Twin-20; 5% formamide, 2.5 mM of MgCl₂, 2.5 U Taq polimerase, 200 mcM of each nucleoside triphosphate, 0.5 mcM of each oligonucleotide primer, 3 mcl of a reference. The amplification program includes: the 1st stage - 94° - 5 min; the 2nd stage - 94° - 60 sec, 55° - 60 sec, 72° - 60 sec (35 cycles); the 3rd stage - 72° - 5 min; 10° - storage. The produced PCR-products in amount 5 mcl are processed by three restrictases - BsfDEl, Btrl, Hpy99l, and restriction products are separated in 3% agarose gel coloured with ethidium bromide.

EFFECT: ensured accurate pattern of restriction analysis of detecting mutations in a rrs MBT gene site responsible for resistance to a group of aminoglycoside preparations.

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Description

The present invention relates to medicine, in particular to a method for diagnosing the sensitivity of M. tuberculosis (MBT) to aminoglycosides (kanamycin, capreomycin and amikacin, etc.). This method allows you to detect mutations in the rrs gene that determine the resistance of the MBT to these drugs by amplifying the corresponding DNA sequences of the MBT gene under study, followed by analysis of the length polymorphism of the obtained restriction fragments after their separation in agarose.

In connection with the increase in the incidence of multidrug-resistant tuberculosis (MBT-MDR), including among patients with newly diagnosed tuberculosis, the question arises of the timely appointment of adequate specific chemotherapy for such patients. In addition, cases of the emergence of resistance of MBT-MDR to fluoroquinolones, one of the most effective reserve-line drugs for the treatment of such patients, have increasingly begun to be recorded. The emergence of such strains created a new problem - the emergence of MBT strains with extensive drug resistance (MBT-XDR), namely, simultaneous resistance to rifampicin, isoniazid, fluoroquinolones and aminoglycosides (Raviglione MC, Smith IM XDR Tuberculosis - implications for Global Public Health. // The New England Journal of Medicine. - 2007, - v. 7, - p. 566-659). To increase the effectiveness of the treatment of such patients, it is necessary to change the chemotherapy regimen using second-line drugs, the most promising of which, in addition to fluoroquinolones, are aminoglycosides (kanamycin, amikacin, capreomycin and viomycin). (Keshavjee S., Gelmanova I.Y et al. Treatment of extensively drug-resistant tuberculosis in Tomsk, Russia: a retrospective cohort study. // Lancet. - 2008, - v.372, - p.1363-1365). In turn, in order to conduct adequate chemotherapy and monitor treatment, it is necessary to quickly determine the drug sensitivity of the office to aminoglycosides.

Obtaining a culture of MBT and determining drug sensitivity in a dense Levenshtein-Jensen medium takes 2-3 months. The use of liquid nutrient media in modern automated systems can reduce the time to obtain culture to 10-18 days. However, currently there are no registered (certified) culture tests on liquid media in the world to analyze the sensitivity of MBT to second-line drugs (fluoroquinolones, aminoglycosides), and determining the sensitivity to them on a dense Levenshtein-Jensen medium increases the analysis time, at least in addition for one month, which does not allow timely adjustments to the chemotherapy regimen.

The development of molecular biological technologies made it possible to study the molecular mechanisms of the resistance of MBT to anti-tuberculosis drugs (PTP) and based on them to put into practice methods that allow determining the drug sensitivity of MBT to PTP within 1-2 days. Thus, using a culture of MBT obtained from a liquid medium and molecular technologies, it is possible to accelerate the obtaining of the result of the sensitivity of MBT to aminoglycosides by 15-20 days.

The target of the action of aminoglycosides is 16S rRNA, upon binding to which the translation process is disrupted with the formation of non-functional proteins, which leads to cell death (bactericidal effect). It has been established that in prokaryotes there are three possible mechanisms for the development of drug resistance to aminoglycosides:

- enzymatic inactivation - the production by bacteria of enzymes that modify antibiotics;

- a decrease in the permeability of the cytoplasmic membrane (violation of cell transport systems);

- modification of the target of action (the receptor protein may be absent or altered as a result of chromosomal mutation).

In the case of the development of MBT resistance to aminoglycosides, the target modification mechanism of the antibiotic action is dominant due to mutations in the rrs gene responsible for the synthesis of 16S rRNA. In addition, it was found that there is cross-resistance of the office to kanamycin, amikacin, capreomycin and viomycin. (Maus CE, Plikaytis V.V., Shinnick TM Molecular analysis of cross-resistance to Capreomycin, Kanamycin, Amikacin and Viomycin in Mycobacterium tuberculosis. // Antimicr. Agents and Chemotherapy. - 2005. - v. 49. - p.3192 -3197). The most common mutation is the replacement of A with G at position 1400 of the rrs gene, which leads to a high level of resistance to kanamycin and amikacin (Alangaden GJ, Kreiswirth BN et al. Mechanism of Resistance to Amikacin and Kanamycin in Mycobacterium tuberculosis. // Antimicr. Agents and Chemoth, - 1998, - v. 42, - p. 1295-1297).

Currently, there are a number of molecular biological approaches to detect mutations in the MBT genes responsible for resistance to anti-TB drugs. The most commonly used methods are conformational polymorphism of single-stranded DNA fragments (SSCP), heteroduplex analysis (GDA), denaturing gradient gel electrophoresis (DGGE), and RNA / RNA heteroduplex analysis (RNA / RNA mismatch assay). All of them are highly sensitive and can detect mutations in 80-95% of cases. But the technological design has a number of drawbacks, such as difficulties in the formulation, in some cases, the use of radioactive tags for detection, work with RNA and RNase, etc. (Foddle R., Losekoot M. Mutation detection by denaturating gradient gel electrophoresis (DGGE) . // Hum. Mutat. - 1994, - v.3, - p. 83-94, Glavac D., Dean M. Optimization of the single-strand conformation polymorphism (SSCP) technique for detection of point mutations. // Hum Mutat. - 1993, - v. 2, - p. 404-414).

An analogue of the present invention is a method for determining mutations using the method of polymorphism of the lengths of restriction fragments in the MBT DNA genes responsible for resistance to chemotherapy drugs in tuberculosis patients (Ahmad S, Mokaddas E, Jaber AA Rapid detection of ethambutol-resistant Mycobacterium tuberculosis strains by PCR-RFLP targeting embB codons 306 and 497 and iniA codon 501 mutations. // Mol Cell Probes. - 2004, - v. 18, - p. 299-306).

The prototype of this invention is the work of Suzuki Y.C., Katsukawa A 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, the authors of which used the restriction analysis method to determine mutations in the rrs MBT gene isolated from Ogawa dense medium. The method proposed in the work consists of several stages:

1. Isolation of DNA from MBT cultures involves buffer treatment followed by DNA purification using phenol-chloroform extraction.

2. Purified MBT DNA in the amount of 10 nanograms is used to amplify a specific region of the rrs gene with a specific pair of primers in the reaction buffer system according to a special amplification program.

3. Precipitation of the PCR product with 3M sodium acetate and alcohol in a ratio of 0.1: 2, followed by dilution in TE buffer.

4. Processing the precipitated PCR product with four restriction enzymes: TaiI, Tsp45I, BstUI and DdeI.

5. Detection of the obtained restriction profiles was carried out after separation on a 4% agarose gel in the presence of SYBR green dye.

This method for determining mutations, primarily according to the conditions of the stages, has a duration (DNA isolation from MBT cultures, precipitation of the resulting amplicons, processing of one MBT DNA sample with four restrictase enzymes) and the use of expensive reagents (SYBR green dye), which is in clinical laboratory conditions becomes not acceptable.

Therefore, in the technique we use, all the steps have been changed:

1) DNA was extracted from MBT cultures in TE buffer (pH 8.0) by heating at 95 ° C for 20 min, which increases the preservation of DNA, accelerates, reduces the cost and simplifies this stage;

2) the composition of the reaction buffer for PCR was changed, namely, the type of buffer, the concentration of triphosphates, magnesium ions, and Taq polymerase. 50 μl of medium contained: 10 mM Tris-HCl pH 8.8; 50 mM KCl; 0.5% tween 20; 5% - formamide, 2.5 mM MgCl ₂ , 2.5 U Taq polymerase, 200 μM of each nucleoside triphosphate, 0.5 μM of each oligonucleotide primer and 3 μl of the sample, which increases the sensitivity and specificity of the PCR reaction;

3) the optimal amplification program was selected, which increases the sensitivity of the method and allows one to determine drug sensitivity on MBT cultures isolated from liquid media, in contrast to the prototype, where MBT cultures from dense media were used. As a result, these changes accelerate the receipt of the result of the sensitivity of the office to aminoglycosides by 15-20 days. The selected conditions are as follows: 1st stage - 94 ° - 5 min; 2nd stage - 94 ° - 60 s, 55 ° - 60 s, 72 ° - 60 s (35 cycles); 3rd stage: 72 ° - 5 min; 10 ° - storage;

4) three restriction enzymes — BstDEI, BtrI, Hpy99I — were selected using the BioEdit v.7.0.1 program; restriction conditions using the obtained amplicons without precipitation (the amplicon volume for restriction is 5 μl), which allows analyzing a larger number of MBT DNA samples and accelerates the preparation result;

5) the separation of restriction products is carried out on a 3% agarose gel stained with ethidium bromide, which reduces the cost of the method.

As a result of the above changes, a clear picture of the restriction analysis of the determination of mutations in the region of the rrs gene of the MBT responsible for resistance to drugs of the aminoglycoside group was obtained.

The drawing shows a photograph of restriction profiles during treatment with restriction enzymes BstDEI, BtrI, Hpy99I of MBT DNA with the detection of mutations in the 1400 codon of the rrs gene.

Claims (1)

A method for diagnosing the sensitivity of Mycobacterium tuberculosis (MBT) to aminoglycosides, including the steps of DNA extraction, production of a polymerase chain reaction (PCR) based on the amplification of DNA of the studied and sensitive strains of MBT, restriction of the PCR product, separation of restriction products in an agarose gel, followed by analysis of restriction profiles characterized in that Mycobacterium tuberculosis DNA is isolated from cultures obtained from a liquid medium by heating for 20 min at 95 ° C in a TE buffer pH 8.0, the selected sequence is amplified using calling the reaction buffer containing 50 μl: 10 mm Tris-HCl pH 8.8; 50 mM KCl; 0.5% tween 20; 5% - formamide, 2.5 mM MgCl ₂ , 2.5 U Taq polymerase, 200 μM of each nucleoside triphosphate, 0.5 μM of each oligonucleotide primer and 3 μl of the sample, according to the optimal amplification program:
1st stage - 94 ° - 5 min;
2nd stage - 94 ° - 60 s, 55 ° - 60 s, 72 ° - 60 s (35 cycles);
3rd stage - 72 ° - 5 min; 10 ° - storage
the resulting PCR products in an amount of 5 μl are treated with three restriction enzymes — BstDEI, BtrI, Hpy99I, and restriction products are separated on a 3% agarose gel stained with ethidium bromide.

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