RU2297456C2 - Diagnosis of mycobacterium tuberculosis strain sensitivity to isoniaside - Google Patents

Abstract

FIELD: medicine, in particular drug screening for tuberculosis treatment.

SUBSTANCE: diagnosis is carried out by determination of tuberculosis mycobacteria sensitivity to isoniaside based on mutation detection in katG, inhA, oxyR/ahpC genes by conformational polymorphism of single-strain fragments and detection mutation in katA gene, which is responsible for abovementioned preparation resistance additionally in 10 % experiences. Also disclosed are primers for DNA of tested katA gene, amplification conditions and electrophoresis conditions. Nucleotide sequences of primer pair are disclosed in specification. Buffer containing in 30 mul Tris-HCl 10 mM, pH 8.8; KCl 50 mM; 0.5 % of Twin 20, formamide 5 %; MgCl 2.0 mM MgCl₂; Tag-polymerase 1.5 U; each mucleoside triphosphate 200 muM; each oligonucleotide primer 0.1-0.15 muM; and sample 3 mul is used for amplification.

EFFECT: improved method for diagnosis of mycobacterium tuberculosis strain sensitivity to isoniaside.

Description

The present invention relates to medicine, in particular to a method for diagnosing the sensitivity of strains of Mycobacterium tuberculosis (MBT) to a first-line drug, isoniazid, for detecting mutations in the kasA gene, which determines resistance to this drug, by analyzing MBT DNA sequences using single-chain conformational polymorphism fragments.

An increase in the incidence of tuberculosis remains one of the most pressing practical public health problems. Against the background of an unfavorable epidemiological situation, an increase in the number of MBT strains with drug resistance to the main anti-tuberculosis drugs (PTP) - isoniazid and rifampicin, which accounts for 95% of cases of multidrug resistance (MDR), is becoming increasingly important.

The main method for determining the sensitivity of MBT to chemotherapeutic agents remains the sowing of bacteriological material on Leveishtein-Jensen medium (L-Y) containing various concentrations of PTP. But it is long and takes 2-3 months, and the results obtained are not relevant for the appointment of adequate treatment to the patient. In recent years, automated systems using liquid media, WASTEC MGIT (Becton Dickinson) and MB / BacT (BioMerieux), which have come into practice in practice, reduce the time needed to obtain the result by 30-40 days, but they do not fundamentally solve this problem and timely determine the sensitivity to PTP, in particular, to isoniazid remains an urgent problem.

One of the most promising areas, at present, is the use of molecular biological methods for determining drug sensitivity to anti-TB drugs. Due to the decoding of the MBT genome, genes have become known whose products are targets for the action of various anti-TB drugs, and the occurrence of mutations in them lead to resistance to these drugs (isoniazid, rifampicin, streptomycin, ethambutol, pyrazinamide). In the case of isoniazid, these are mainly mutations in four genes: kat G, inh A, ahp C, and kas A. It is known from the literature that the activity of genes is interconnected: resistance to kat G increases, decreases to inh A, activity increases non-guimine peroxidase for which ahp C is responsible (Ortiz de Montellanop R., Hillas P., Zhang Z., Wilks A. Oxidative stress drag resistance in M. tuberculosis. Rev tarm e bioguim Univ. Sao Paulo. 1998. 34. No. 1 p. 53). Therefore, the analysis of the work of each of the genes is important for understanding the mechanism of resistance of the office to isoniazid. It is known that about 10-15% of MBT strains have a mutation in the kas A gene. The detection of mutations in the kat G, inh A, and ahp C genes determines isoniazid sensitivity for 80% of MBT strains (previous invention No. 2200323), i.e., determination of sensitivity by kas A provides additional information on the studied strain.

A wide range of methods has been developed for the detection of bacterial mutations. These are: allele-specific polymerase chain reaction (PCR), chain extension with labeled dideoxyribonucleoside triphosphate, analysis of restriction fragment polymorphism, sequencing, dideoxy-fingerprinting, imperfect duplex method, structural-specific nuclease cleavage method, “reverse” hybridization of DNA immobilized immobilized DNA hybridobases on microchips, the method of conformational polymorphism of single-chain fragments. Each of the presented methods has its own advantages and disadvantages, but these methods are mainly used at present in research laboratories to solve theoretical problems (Delgado MB, Telenti A. Detection of Fluoroquinolone Resistance Mutation in M. tuberculosis. In PCR Protocols for Emerging Infections Diseases. 1996, ASM press, Washington, p. 138-143; Telenti A., Imboden P., Marchesi F., et al. Detection of rifampin resistance mutation in M. tuberculosis. Lancet. 1993.341, p. 647-650; Caugant DA, Sandven P., Eng J., Jeque T. Detection of rifampin resistance among of Mycobacterium tuberculosis from Mozambigue. 1995, 4, p. 321-325).

An analogue of this invention is a method for determining mutations in the MBT DNA genes responsible for isoniazid resistance in tuberculosis patients (Victor TC, Jordaan AM Detection of mutation in drug resistance genes of M. tuberculosis by a dot-blot hybridization strategy. 1999. Int. J . Tuberc and Lung Disease. 79 (6), p. 343-348. Bahrmand A., Bakaev V. Rapid detection of multidrug-resistant tuberculosis using molecular genetic metods. 1998. Int. J. Tuberc and Lung Disease. 2, 11 , p. 346; Gonzales N., Torres J., Aznar J., Polomares JC Molecular analysis of rifampin and isoniazid resistance of M. tuberculosis clinical isolates in Seville. 1999. Tubercle and Lung Disease. 79, 3, p. 187- 190 .; Pretorius GS, van Helden PD, Sirgel F., Eisenach KD Mutation in katG Gene Seguences in Isoniazid-Resistant Clinical Isolates of M. tuberculosis are Rare. Antimicro b. Agents Chemother. 1995, 39, 10, p. 2276-2281).

These authors studied isoniazid resistance associated with mutations in the katG, inhA, and ahpC genes. Each author used his amplification conditions for each gene under study, which significantly increased the amount of work.

The prototype of this invention is the previously proposed complex that combines a polymerase chain reaction with further analysis of amplicons by the method of conformational polymorphism of single-chain fragments (PCR-SSCP) to determine mutations in the 3 main M. tuberculosis genes: katG, ahpC, inhA, responsible for isoniazid resistance (previous invention No. 220032).

This set of methods consists of several stages:

The studied DNA sequence of the studied object is cloned by PCR.

The PCR product undergoes thermal denaturation in the presence of double helix destabilizing agents, for example formamide, and is transferred to ice.

The mixture is applied to a polyacrylamide gel and separated by high voltage electrophoresis.

Staining of the gel is carried out using a dye of nitric acid silver or Sybr Green 2.

The initial sequences have different electrophoretic mobility associated with the geometric characteristics of the supramolecular structures formed by this sequence. The resulting pattern is specific to the sequence being studied, and in the case of replacing one nucleotide in the chain, the pattern obtained by electrophoresis changes, i.e., the strands of amplified denatured sequences from the studied DNA sections diverge at different distances from each other. The degree of discrepancy between the denatured strands is detected by gel staining.

Thus, this method is quite accessible (polymerase chain reaction method, electrophoresis technique is now used in all biochemical laboratories), specific (work is carried out directly with bacterial DNA) and quite sensitive (because single cell DNA is detected by polymerase chain reaction).

The differences of our proposed method from the previous one are as follows. A study of sensitivity to isoniazid is carried out on another kasA gene, which is also involved in resistance to this drug. For this gene, pairs of primers were selected, as well as an amplification program. After electrophoresis on an electrophoregram, mutations in the kasA gene can be detected. Thus, within two days, it is possible to determine the sensitivity to isoniazid by the fourth main gene and thereby ensure the probability of detecting mutations responsible for isoniazid resistance, additionally in another 10-15% of cases.

To solve the problem, conditions were first developed for each of the stages of the method:

- for the synthesis of MBT DNA of the kasA gene from several pairs of primers, the following were selected:

KasA gene: 1st - 5GGC GAC GCG GAT GGC GTT 3

2nd - 5TCG AGA CGG AGG AGC ACG CCA A 3

- the composition of the amplification buffer was selected, where the important components are the type of buffer, the concentration of triphosphates, the concentration of magnesium ions, so that the concentration of magnesium ions in the composition of the reaction medium for the kasA gene was changed. 30 μl of medium contained: 10 mM Tris-HCl pH 8.8; 50 mM KCl; 0.5% tween 20; 5% - formamide, 2.0 mM MgCl ₂ , 1.5 U Taq polymerase, 200 μM of each nucleoside triphosphate, 0.1 - 0.15 μM of each oligonucleotide primer and 3 μl of sample;

- the amplification mode was selected, varying the annealing temperatures for a pair of primers, the time of each cycle and the number of cycles. The amplification mode was selected in such a way that the conditions for sufficient synthesis of the necessary MBT DNA fragments of the kasA gene coincided with the amplification program for the katG, ahpC, and inhA genes (previous invention No. 2200323), which greatly simplified the work of detecting mutations in all four genes. The selected conditions are as follows: 1st stage: 95 ° - 5 min, 60 ° - 2 min; 2nd stage: 72 ° - 1 min, 95 ° - 40 sec, 60 ° - 1 min (35 cycles); 3rd stage: 72 ° - 10 min;

- conditions were selected for studying the conformational polymorphism of single-chain amplicons obtained in the previous step, namely, the conditions for the denaturation of the resulting amplicons (the ratio of amplicons and the denaturing solution, the time of denaturation), the composition of the polyacrylamide gel (percentage of polyacrylamide with glycerin and the composition of the buffer), separation conditions ( voltage, electrophoresis time, temperature), allowing to increase the separation efficiency of single-stranded fragments of the studied amplicons. The selected conditions are as follows: denaturation is carried out at 95 ° C for 10 min, 3 μl of the sample and 3 μl of denaturing solution are mixed for denaturation, the separation of the denatured amplicons is carried out in 8% polyacrylamide gel with 5% glycerol at 8 ° C, voltage 450 V, 5 hours in TVE buffer.

Electrophoregrams were stained with Sybr Green 2, according to the recommendations of the manufacturer of this reagent.

Detection of results. After staining, the electrophoregram is placed under UV (transilluminator), the wavelength is 254 nm. Colored denatured amplicons are two white luminous stripes for each sample. To determine mutations in the gene, along with amplicons of the test sample, amplicons of DNA of the MBT sensitive strain are also examined. If the MBT strain is resistant to isoniazid, the distance between the denatured strands of amplified DNA fragments of resistant strains differs from the distance between the strands of amplified DNA fragments of the sensitive strain, i.e., primers, amplification and detection conditions were selected according to the previously proposed invention for the detection of mutations in katG, ahpC genes , inhA so that under the same conditions of amplification and detection, from one sample, using only different primers, it is possible to determine the sensitivity to niazidu to identify mutations in the four genes, whose presence is typical for 90% MBT strains.

The proposed method for detecting the minimum amount of MBT DNA in the sample showed that using the proven method it is possible to identify DNA equivalent to the DNA of single MBT cells. Studies to identify isoniazid resistance were performed on MBT DNA obtained from clinical samples and clinical isolates from these samples grown on liquid media and L-Y medium. Samples were obtained from patients with different forms of tuberculosis being treated at the ISTC BT.

Claims (1)

A method for diagnosing the sensitivity of Mycobacterium tuberculosis to isoniazid, including setting up a polymerase chain reaction (PCR) using primers and amplification buffer, denaturing the obtained amplicons, detecting mutations in the three main genes katG, ahpC, inhA by the method of conformational polymorphism of single-chain fragments by diagnosis and diagnosis their electrophoretic mobility in a polyacrylamide gel, characterized in that mutations in the kasA gene are additionally detected using a pair of right-handed PCRs mer 5 'GGC GAC GCG GAT GGC GTT 3' and 5 'TCG AGA CGG AGG AGC ACG CCA A 3', and amplification buffer containing 30 .mu.l of 10 mM Tris-HCl pH 8.8; 50 mM KCl; 0.5% tween 20; 5% - formamide, 2.0 mM MgCl ₂ , 1.5 U Tag polymerase, 200 μM of each nucleoside triphosphate, 0.1-0.15 μM of each oligonucleotide primer and 3 μl of the sample.