RU2806581C1 - Method for diagnosing helicobacter pylori infection and its resistance to clarithromycin and levofloxacin - Google Patents

Abstract

FIELD: molecular biology.

SUBSTANCE: method for detecting Helicobacter pylori infection and its resistance to clarithromycin and levofloxacin in a biopsy sample of the gastric mucosa of a patient is described. The method includes carrying out a polymerase chain reaction using a PCR reaction solution and primers, purifying the PCR product, sequencing reaction, purifying the sequencing reaction product, detecting and analyzing the result, while the test system contains a set of reagents for PCR, buffer, MgCl₂, dNTP, Taq polymerase, DNA sample and primer set.

EFFECT: increasing the accuracy of diagnosing Helicobacter pylori infection and its resistance to clarithromycin and levofloxacin.

1 cl, 6 dwg, 3 ex

Description

The invention relates to the field of molecular genetic diagnostics, molecular biology and biotechnology and can be used to diagnose the presence of Helicobacter pylori (HP) infection and its resistance to clarithromycin and levofloxacin.

Well-known methods for diagnosing CP and its antibiotic resistance are molecular genetic methods, in particular the Sanger sequencing method. The difference between this method and other methods is its accuracy. The method involves reading the nucleotide sequence of gene sections specific to all HP strains, which are not found in other types of bacteria, as well as the nucleotide sequence of gene sections in which mutations determine its antibiotic resistance. Resistance to clarithromycin is associated with variants 2142A>C, 2142A>G and 2143A>G in the 23S rRNA gene, and resistance to levofloxacin is associated with variants 87 (259A>T, 261T/C<G/A) and 91 (271G>A , 271G>T, 272A>G) codons of the gyrA gene [1 - Monica Oleastro et al. Real-Time PCR Assay for Rapid and Accurate Detection of Point Mutations Conferring Resistance to Clarithromycin in Helicobacter pylori // Journal of clinical microbiology, Vol. 41, No. 1. - 2003, p. 397-4025].

This method is accepted as analogous.

There is a known method for diagnosing CP and its antibiotic resistance. The invention is a chip for detecting the drug resistance gene of Helicobacter pylori to clarithromycin, a method for its production and use. The technical solution consists of probes placed on a carrier. The present invention realizes the fast and simple detection of polymorphisms of the 23SrRNA 2142, 2143 and 2182 genes directly from a sample of the gastric mucosa, no bacterial culture is required [2 - patent CN 200410073454, published 2007-01-17 “Chip for detecting polymorphism of the Helicobacter pylori resistance gene to clarithromycin "]. This method is adopted as a prototype.

A significant disadvantage of this technical solution, firstly, is that this method can only determine resistance to clarithromycin; secondly, for its implementation in clinical practice, it is necessary to have special equipment for detecting chips and the constant purchase of the chips themselves, which is not economically profitable for routine work. In addition, the method of hybridization on a substrate has such a negative side as nonspecific hybridization and, as a consequence, nonspecific fluorescence and false results.

The goal is to improve the accuracy of diagnosis of Helicobacter pylori infection and its resistance to clarithromycin and levofloxacin.

The technical result is

a method for diagnosing Helicobacter pylori infection and its resistance to clarithromycin and levofloxacin in a biopsy sample of the patient’s gastric mucosa, including DNA extraction, performing a PCR reaction using a test system, analyzing the resulting products on a DNA analyzer, characterized in that a reaction solution for PCR is used and primers SEQ ID NO1: 5'-TAAATACCGACCTGCATGAATG-3', SEQ ID NO 2: 5'-CTCCATAAGAGCCAAAGCCC-3', SEQ ID NO 3: 5'-GGAAACCCTGAAGC AGC A ACG-3', SEQ ID NO 4: 5' -CGCCTTCGC AATGAGTATTCCT-3', SEQ ID NO 5: 5'-ATC AT AGGGCGTGCTTT ACC-3', SEQ ID NO 6: 5'-AAAGGTTAGGCAGACGGCTT-3', PCR product purification, sequence reaction, sequence reaction product purification, detection and analysis of the result obtained, while the test system contains a set of reagents for PCR, a buffer, MgCl2, dNTP, Taq polymerase, a DNA sample and a set of primers, SEQ ID NO 1: 5'-TAAATACCGACCTGCATGAATG-3', SEQ ID NO 2: 5'-CTCCATAAGAGCCAAAGCCC-3', SEQ ID3: 5'-GGAAACCCTGAAGC AGC A ACG-3', SEQ ID NO 4: 5'-CGCCTTCGC AATGAGTATTCCT-3', SEQ ID NO 5: 5'-ATC ATAGGGCGTGCTTTACC-3', SEQ ID NO 6: 5'-AAAGGTTAGGCAGACGGCTT-3'.

The method according to claim 1 for detecting Helicobacter pylori resistance to clarithromycin and levofloxacin, characterized in that the primers are used: SEQ ID 1-6: SEQ ID 1: 5'-TAAATACCGACCTGCATGAATG-3', SEQ ID2: 5'-CTCCATAAGAGCCAAAGCCC-3' , SEQ ID3: 5'-GGAAACCCTGAAGC AGC AACG-3', SEQ ID4: 5'-CGCCTTCGCAATGAGTATTCCT-3', SEQ ID5: 5'-ATCTAGGGCGTGCTTTACC-3', SEQ ID6: 5'-AAAGGTTAGGCAGACGGCTT-3'.

The reference nucleotide sequences of the S23, GyrA, and S16 Helicobacter pylori gene regions are presented.

A list of nucleotide sequences (primers) is presented.

In FIG. Figure 1 shows the alignment of a fragment of the Helicobacter pylori nucleotide sequence found in patient No. 1 with the reference sequence of the GyrA rRNA gene (A) and the alignment of a fragment of the Helicobacter pylori nucleotide sequence found in patient No. 1 with the reference sequence of the Helicobacter pylori S23 rRNA gene (B).

In FIG. Figure 2 shows chromatograms of the nucleotide sequence of the S23 rRNA (A) and GyrA rRNA (B) genes of Helicobacter pylori in patient No. 1.

In FIG. Figure 3 shows the alignment of a fragment of the Helicobacter pylori nucleotide sequence found in patient No. 2 with the reference sequence of the GyrA rRNA gene (A) and the alignment of the Helicobacter pylori nucleotide sequence found in patient No. 2 with the reference sequence of the S23 rRNA gene (B).

In FIG. Figure 4 shows chromatograms of the nucleotide sequence of the GyrA rRNA (A), S23 rRNA (B) Helicobacter pylori genes in patient No. 2.

In FIG. Figure 5 shows the alignment of the nucleotide sequence of Helicobacter pylori found in patient No. 3 with the reference sequences of the GyrA rRNA (A) and S23 rRNA (B) genes.

In FIG. Figure 6 shows chromatograms of the nucleotide sequence of genes and GyrA rRNA (A) and S23 rRNA (B) of Helicobacter pylori in patient No. 3.

The method is carried out as follows.

Currently, the Russian Federation is characterized by a high prevalence of CP infection. According to the literature, more than 112 million people are infected with CP. The International Agency for Research on Cancer has classified CP as a Group 1 carcinogen. In the Maastricht V recommendations, eradication therapy is a strategy to reduce the risk of developing erosive and ulcerative lesions of the mucous membrane of the stomach and duodenum, and also helps to prevent precancerous conditions [3 - Stepanov Yu.M., Budzak I.Ya. Maastricht Consensus - 5: analytical review of provisions // Gastroenterology, v. 51, no. 1. - 2017., pp. 36-45]. Like other bacteria, HP has regional patterns of resistance, and the number of antibiotic-resistant strains is growing due to the widespread use of drugs, which reduces the effectiveness of eradication therapy. For example, the literature describes that resistance to clarithromycin in the Netherlands is only 5.6%, while resistance of CP to this antibiotic in Austria reaches 35.4%. The metronidazole resistance rate in Beijing was 63.9%, and in the Southeast Coast of China it was 95.4%. In Russia, the level of resistance to Clarithromycin is 10%, to Levofloxacin - 20%, Metronidazole - 34%. [4 - Andreev D.N., Maev I.V., Kucheryavyi Yu.A. Resistance of Helicobacter pylori in the Russian Federation: meta-analysis of studies over the past 10 years // Therapeutic archive, 11, 2020. - pp. 24-30. Doi: 10.2644/00403660.2020.11.000795]. Resistance to HP antibiotics is increasing worldwide, highlighting the need for more rapid detection of resistance before prescribing treatment regimens for HP [5 - A. Savoldi, E. Carrara, D.Y. Graham, M. Conti, E. Tacconelli Prevalence of Antibiotic Resistance in Helicobacter pylori: A Systematic Review and Metaanalysis in World Health Organization Regions // Gastroenterology. - 2018. 155(5): 1372-1382].

Recent guidelines suggest that first-line therapy for eradicating HP infection is a standard triple regimen consisting of a proton pump inhibitor (PPI), clarithromycin, and amoxicillin. As an alternative first-line eradication therapy, the classic four-component regimen based on bismuth tripotassium dicitrate or quadruple therapy without bismuth drugs, which includes PPIs, amoxicillin, clarithromycin and metronidazole, can be used. Quadruple therapy with bismuth tripotassium dicitrate is also used as the main second-line regimen when standard triple therapy is ineffective. Other second-line treatment regimens include PPIs, levofloxacin, and amoxicillin. Third-line therapy is selected individually based on determining the sensitivity of CP to antibacterial drugs. The results of some studies indicate that in order to overcome resistance to clarithromycin, the addition of bismuth tripotassium dicitrate to classical therapy is recommended. Also, among the modification methods that can improve the effectiveness of eradication therapy for H. pylori infection, it should be noted that the duration of treatment is increased to 14 days, the choice of a more modern PPI or an increase in the dose of PPI, the addition of bismuth tripotassium dicitrate or a probiotic. It is important that the appointment of eradication therapy, in case of detection of H. pylori, is especially important as an effective measure for the prevention of gastric cancer [3 - Stepanov Yu.M., Budzak I.Ya. Maastricht Consensus - 5: analytical review of provisions // Gastroenterology, v. 51, no. 1. - 2017., pp. 36-45].

As a result of antibacterial therapy, a number of patients experience side effects, such as: diarrhea, nausea, vomiting, bacterial overgrowth syndrome, pseudomembranous colitis, the development of allergic reactions and others. The development of side effects forces patients to prematurely interrupt eradication therapy, which in turn is a factor in increasing bacterial resistance to antibiotics and subsequently reduces the effectiveness of therapy. Thus, a number of difficulties arise when prescribing treatment regimens for infection. Determining resistance in a short time will allow doctors to prescribe eradication therapy in a personalized manner, which will improve drug tolerability and increase patient compliance with treatment.

Today, the following methods for diagnosing CP infection are used: the histological method (when taking a biopsy) - despite the high sensitivity of the method, its accuracy may be affected by the amount of biopsy material and the location of their collection, from the point of view of uneven distribution over the gastric mucosa. During the rapid urease test, biopsy material from the gastric mucosa is placed in a special solution. In the presence of CP, due to the urease produced, the solution will change color to crimson. The result is available in 15-30 minutes. Even though the test is quite fast, it still has a number of disadvantages. Both false positive and false negative results are possible. False-positive results are possible due to the fact that other bacteria that inhabit the gastric mucosa may also produce urease and color the test solution. False-negative results can occur with a low degree of contamination of the gastric mucosa with CP. The serological method (presence of antibodies to CP in the blood) is not very informative in patients with a weak immune response, early stage of infection, because IgG antibodies appear no earlier than a month after infection. Despite the fact that the level of antibodies decreases during successful eradication, the serological reaction remains positive for a number of years, so this method cannot be used to monitor the effectiveness of anti-Helicobacter therapy. Analysis of feces for the CP antigen is a fairly accurate method, but with constipation and difficult bowel movements, the CP membrane may be destroyed. The 13C-urease breath test is a fairly accurate test, however, false negative results can be observed in patients who have undergone gastrectomy, as well as in patients with a significant decrease in gastric secretion. False-positive tests may be due to the breakdown of urea by bacteria in the mouth or by bacteria containing urease in the stomach. Another disadvantage of the method is its high cost. The bacteriological method is based on identifying the pathogen by culture from a biopsy of the gastric mucosa. The bacteriological method allows you to cultivate CP using biopsies of the gastric mucosa, identify the bacterium, study its morphological, biochemical and biological properties, and allows you to study the pathogenicity factors of CP. The culture method has the highest specificity, but the sensitivity of the method is reduced due to the fact that special care is required when handling samples of the gastric mucosa. The method is quite expensive, very labor-intensive and rarely used in routine clinical practice [6-Bordin D.S. and others. “Methods for diagnosing Helicobacter pylori infection.” Guidelines. - Moscow, 2019].

A method for diagnosing CP infection and its resistance to clarithromycin and levofloxacin includes DNA isolation, carrying out a PCR reaction using a test system, analysis of the resulting products on a DNA analyzer, additionally introducing the following features: primers specific to various CP strains flanking the target point area S16 rRNA genes (SEQ ID NO 3 - 5'-GGAAACCCTGAAGCAGCAACG-3', SEQ ID NO 4 - 5'-CGCCTTCGCAATGAGTATTCCT-3') - for diagnosing the presence of CP infection, S23 rRNA (SEQ ID NO 1 - 5'-TAAATACCGACCTGCATGAATG- 3', SEQ ID NO 2 - 5'-CTCCATAAGAGCCAAAGCCC-3') - for diagnosing resistance to clarithromycin, GyrA rRNA (SEQ ID NO 5-5'-ATC ATAGGGCGTGCTTTACC-3', SEQ ID NO 6 - 5' - A AAGGTTAGGC AG ACGGCTT-3') - for diagnosing resistance to levofloxacin.

The method is illustrated by the following figures:

Fig. 1 alignment of a fragment of the Helicobacter pylori nucleotide sequence found in patient No. 1 with the reference sequence of the GyrA rRNA gene (A) and alignment of a fragment of the Helicobacter pylori nucleotide sequence found in patient No. 1 with the reference sequence of the Helicobacter pylori S23 rRNA gene (B).

Fig. 2 chromatograms of the nucleotide sequence of the S23 rRNA (A) and GyrA rRNA (B) genes of Helicobacter pylori in patient No. 1.

Fig. 3 alignment of a fragment of the Helicobacter pylori nucleotide sequence found in patient No. 2 with the reference sequence of the GyrA rRNA gene (A) and alignment of the Helicobacter pylori nucleotide sequence found in patient No. 2 with the reference sequence of the S23 rRNA gene (B).

Fig. 4 chromatograms of the nucleotide sequence of the genes GyrA rRNA (A), S23 rRNA (B) Helicobacter pylori in patient No. 2.

Fig. 5 alignment of the nucleotide sequence of Helicobacter pylori found in patient No. 3 with the reference sequences of the GyrA rRNA (A) and S23 rRNA (B) genes.

Fig. 6 chromatograms of the nucleotide sequence of genes and GyrA rRNA (A) and S23 rRNA (B) Helicobacter pylori in patient No. 3.

The presence of infection is determined by the presence of the S16 rRNA gene amplification product. Next, the resulting amplicons are subjected to a sequence reaction, followed by separation of the sequence reaction products by capillary gel electrophoresis on a genetic analyzer. Determine the presence or absence of nucleotide substitutions A2142A>G, A2142A>C and A2143A>G in the 23S rRNA gene, nucleotide substitutions 259A>T, 261T/C>G/A in the 87th codon of the gene and 271G>A, 271G>T, 272A> G in codon 91 of the GyrA rRNA gene.

Thus, both in the presence of a mutation and without it, the presence or absence of CP infection will be diagnosed with 100% accuracy (the nucleotide sequence of the gene is directly “read”) and, if present, the resistance of CP to clarithromycin and levofloxacin will be determined.

To implement the claimed method, a test system is proposed containing a reaction mixture for a PCR reaction and primers for amplification of a DNA sequence, into which the following new features have been introduced:

1. Buffer for PCR reaction with MgC12 - 1.5 ml.

2. dNTP mixture (dATP, dGTP, dCTP, dTTP) - 200 µM.

3. Taq polymerase - 1000-5000 units. activity.

4. DNA control samples (positive control sample, negative control sample).

5. Primers (SEQ ID 1-6):

SEQ ID NO 1: 5'-TAAATACCGACCTGCATGAATG-3' (straight)

SEQ ID NO 2: 5'-CTCCATAAGAGCCAAAGCCC-3' (reverse)

SEQ ID NO 3: 5'-GGAAACCCTGAAGCAGCAACG-3' (straight)

SEQ ID NO 4: 5'-CGCCTTCGCAATGAGTATTCCT-3' (reverse)

SEQ ID NO 5: 5' - PBX ATAGGGCGTGCTTTACC-3' (direct)

SEQ ID NO 6: 5'-AAAGGTTAGGCAGACGGCTT-3' (reverse)

To solve this problem, an individual design of primers was made using the Oligo program.

The design of the primers was carried out based on the following principles: the annealing region of the primers should be outside the mutation zones within the limits of species specificity; GC composition 40-60%; primers should have very similar melting temperatures in the range of 60-66°C; The primer sequence must be free of stable secondary structures - hairpins and dimers. To exclude amplification of off-target loci, the uniqueness of each primer was confirmed by searching the sequenced genome database http://wvvw.ensembl.org.

The specificity of the test was confirmed by the study of other microorganisms: Enterococcus faecalis, Proteus vulgaris, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus viridans, Pseudomonas aeruginosa, Candida albicans, Haemophilus influenzae, Campylobacter jejuni, Herpes Simpex virus, Varicella Zo oster, Cytomegalovirus, Epstein Bar viruses.

The method based on the test system is carried out as follows:

DNA is extracted from a biopsy of the gastric mucosa obtained during esophagogastroduodenoscopy.

The isolated DNA is then subjected to polymerase chain reaction using the test system described above. Polymerase chain reaction (PCR) is performed on thermal cyclers in a reaction mixture with a volume of 25 μl, 67 mM Tris-HCl (pH = 8.8), 2.5 mM MgCl2, 1 ng of genomic DNA, 10 pM each of forward and reverse primers, 200 µM dATP, dGTP, dCTP, dTTP and 1 unit of active Taq polymerase. The PCR mode is as follows: 1) denaturation 95°C - 5 minutes; 35 cycles: 2) denaturation 95°C - 30 sec.; primer annealing: 60-66°C - 30 sec.; elongation 72°C - 1 min.; 3) final elongation 72°C - 5 min.

Next, detection of amplification products is carried out by horizontal electrophoresis in a 2% agarose gel. The presence of amplification products for the S16, GyrA, S23 rRNA genes indicates the presence of CP infection. If CP is detected, then the amplifier generated by PCR is purified using columns and a sequence reaction is carried out using dideoxynucleotides (terminators) by thermal cycling: denaturation 96°C - 1 min.; 25 cycles: denaturation 96°C - 10 sec.; primer annealing 60°C - 5 sec.; elongation - 60°C - 4 min. Next, the amplifier is purified by any method suitable for purifying a sequence product, followed by separation of nucleotides by capillary gel electrophoresis on a genetic analyzer.

The method is confirmed by the following examples.

Example 1.

Patient T. aged 50 years. Complaints of pain in the right hypochondrium, epigastric discomfort. From the anamnesis it is known that the above complaints have been bothering me for about one and a half years. According to esophagogastrodulodenoscopy (EGDS) - antral gastropathy. According to IgGr serology, CP was detected. According to EGD data from 2021 - cardia insufficiency. Diffuse gastritis. As a result of endoscopy, a sample of the gastric mucosa was taken from the patient. DNA was isolated using a standard method for extracting DNA from human tissue.

Next, a PCR reaction was carried out according to the following protocol: deionized water - 12.5 µl; buffer - 5 µl; MgC12 - 2.5 µl; primers - 1 μl of forward and reverse (SEQ ID NO 1-6: SEQ ID 1: 5'-TAAATACCGACCTGCATGAATG-3', SEQ ID NO 2: 5'-CTCCATAAGAGCCAAAGCCC-3', SEQ ID NO 3: 5'-GGAAACCCTGAAGCAGCAACG -3', SEQ ID NO 4: 5'-CGCCTTCGC AATGAGTATTCCT-3', SEQ ID NO 5: 5'-ATCATAGGGCGTGCTTTACC-3', SEQ ID NO 6: 5'-AAAGGTTAGGCAGACGGCTT-3', Appendix 1); dNTP - 0.5 µl; Taq polymerase - 0.5 µl; DNA - 2 µl. PCR mode: 1) denaturation 95°C - 5 min.; 35 cycles: 2) denaturation 95°C - 30 sec.; primer annealing: 60-66°C - 30 sec.; elongation 72°C - 1 min.; 3) final elongation 72°C - 5 min.

Next, the presence of amplification products was checked using horizontal electrophoresis in a 2% agarose gel. The luminescence of bands was recorded in all wells containing the PCR product, which indicates the presence of CP infection. The amplification was purified on columns according to the manufacturer's instructions.

Next, a sequence reaction was performed according to the following protocol: deionized water - 5.5 μl; terminator - 0.5 µl; 5x buffer - 1 µl; primer - 1 µl. direct or reverse (SEQ ID NO 1-6: SEQ ID NO 1: 5'-TAAATACCGACCTGCATGAATG-3', SEQ ID NO 2: 5'-CTCCATAAGAGCC AAAGCCC-3', SEQ ID NO 3: 5'-GGAAACCCTGAAGC AGC AACG- 3', SEQ ID NO 4: 5'-CGCCTTCGC AATGAGT ATTCCT-3', SEQ ID NO 5: 5'-ATCATAGGGCGTGCTTTACC-3', SEQ ID NO 6: 5'-AAAGGTT AGGC AG ACGGCTT-3', Appendix 1 ); PCR product - 2 µl. Sequence reaction mode: denaturation 96°C - 1 min.; 25 cycles: denaturation 96°C - 10 sec.; primer annealing 60°C - 5 sec.; elongation - 60°C - 4 min.

Next, the amplifier was purified using the sorbent method. Subsequent separation of nucleotides was carried out by capillary gel electrophoresis on a genetic analyzer. To determine nucleotide substitutions at target points, the obtained sequences are aligned with the reference sequence. Alignment of sequences of the DNA sample of patient No. 1 with reference sequences is presented in Fig. 1.

As a result of the study, the patient was found to have a CP infection - S16+, no resistance to clarithromycin was found - S23 (2142A, 2143A) - wild type (wt), resistance to levofloxocin was found - GyrA 87 codon (259A, 261T) - wild type (wt); GyrA codon 91 (27IT - mutation (mut), 272A - wild type (wt) (Fig. 2).

Example 2.

Patient S. aged 57 years. There is a history of cancer of the mucous membrane of the alveolar process of the left upper jaw. A biopsy of a neoplasm of the mucous membrane of the alveolar process of the left upper jaw was performed, according to a histological examination performed in 2021 - squamous cell keratinizing carcinoma of a high degree of differentiation of the mucous membrane of the upper jaw. Endoscopy is recommended. According to IgGr serology, CP was detected.

As a result of endoscopy, a sample of the gastric mucosa was taken from the patient. DNA was isolated using a standard method suitable for extracting DNA from human tissue.

Next, a PCR reaction was carried out according to the following protocol: deionized water - 12.5 µl; buffer - 5 µl; MgCl2 - 2.5 µl; primers - 1 µl. forward and reverse (SEQ ID NO 1-6: SEQ ID NO 1: 5'-TAAATACCGACCTGCATGAATG-3', SEQ ID NO 2: 5'-CTCC AT AAGAGCC AAAGCCC-3', SEQ ID NO 3: 5'-GGAAACCCTGAAGC AGC AACG-3', SEQ ID NO 4: 5'-CGCCTTCGC AATGAGTATTCCT-3', SEQ ID NO 5: 5'-ATCATAGGGCGTGCTTTACC-3', SEQ ID NO 6: 5'-AAAGGTTAGGCAGACGGCTT-3', Appendix 1); dNTP - 0.5 µl; Taq polymerase - 0.5 µl; DNA - 2 µl. PCR mode: 1) denaturation 95°C - 5 min.; 35 cycles: 2) denaturation 95°C - 30 sec.; primer annealing: 60-66°C - 30 sec.; elongation 72°C - 1 min.; 3) final elongation 72°C - 5 min.

Next, the presence of amplification products was checked using horizontal electrophoresis in a 2% agarose gel. The luminescence of bands was recorded in all wells containing the PCR product, which indicates the presence of CP infection. The amplification was purified on columns according to the manufacturer's instructions.

Next, a sequence reaction was performed according to the following protocol: deionized water - 5.5 μl; terminator - 0.5 µl; 5x buffer - 1 µl; primer - 1 µl forward or reverse (SEQ ID 1-6: SEQ ID NO 1: 5'-TAAATACCGACCTGCATGAATG-3', SEQ ID NO 2: 5'-CTCC AT AAGAGCC AAAGCCC-3', SEQ ID NO 3: 5 '-GGAAACCCTGAAGC AGC AACG-3', SEQ ID NO 4: 5'-CGCCTTCGC AATGAGT ATTCCT-3', SEQ ID NO 5: 5' - ATC ATAGGGCGTGCTTT ACC-3', SEQ ID NO 6: 5'-AAAGGTTAGGCAGACGGCTT-3 ', Appendix 1); PCR product - 2 µl. Sequence reaction mode: denaturation 96°C - 1 min.; 25 cycles: denaturation 96°C - 10 sec.; primer annealing 60°C - 5 sec.; elongation - 60°C - 4 min.

Next, the amplifier was purified using the sorbent method. Subsequent separation of nucleotides was carried out by capillary gel electrophoresis on a genetic analyzer. To determine nucleotide substitutions at target points, the obtained sequences were aligned with the reference sequence. Alignment of the sequences of the DNA sample of patient No. 2 with reference sequences is presented in Fig. 3.

As a result of the study, the patient was diagnosed with CP infection - S16+, no resistance to clarithromycin was detected - S23 (2142A, 2143 A) - wild type (wt), resistance to levofloxocin was not detected - GyrA 87 codon (259A, 261C) - wild type (wt ); GyrA codon 91 (271G, 272A) - wild type (wt) (Fig. 4).

Example 3.

Patient K. aged 60 years. Diagnosis: gastritis. Complaints of epigastric pain radiating to the back, chest discomfort, frequent constipation. History of the disease: the patient has been suffering from constipation for a long time, periodically with errors in nutrition, pain in the epigastrium with radiation to the back. CT scan of ABP dated November 30, 21 - there are no signs of space-occupying formations, no stones. CEA 4.89 ng/ml. Twice successful eradication of CP, positive C13 urease endoscopy dated 04/17/21 - condition after fundoplication, antral gastritis CS dated 04/17/21 - no pathology was detected. The patient has been suffering from constipation for a long time and uses various groups of laxatives. According to endoscopy data from 2022, single venectasis of the middle third of the esophagus. Epithelial formation of the esophagus (verification based on the results of a morphological study). The condition after fundoplication in 2010 was superficial gastritis.

As a result of endoscopy, a sample of the gastric mucosa was taken from the patient. DNA was isolated using a standard method suitable for extracting DNA from human tissue.

Next, a PCR reaction was carried out according to the following protocol: deionized water - 12.5 µl; buffer - 5 µl; MgCl2 - 2.5 µl; primers - 1 µl. forward and reverse (SEQ ID NO 1-6: SEQ ID NO 1: 5'-TAAATACCGACCTGCATGAATG-3', SEQ ID NO 2: 5'-CTCC AT AAGAGCC AAAGCCC-3', SEQ ID NO 3: 5'-GGAAACCCTGAAGC AGC AACG-3', SEQ ID NO 4: 5'-CGCCTTCGC AATGAGTATTCCT-3', SEQ ID NO 5: 5'-ATCATAGGGCGTGCTTTACC-3', SEQ ID NO 6: 5'-AAAGGTTAGGCAGACGGCTT-3', Appendix 1); dNTP - 0.5 µl; Taq polymerase - 0.5 µl; DNA - 2 µl. PCR mode: 1) denaturation 95°C - 5 min.; 35 cycles: 2) denaturation 95°C - 30 sec.; primer annealing: 60-66°C - 30 sec.; elongation 72°C - 1 min.; 3) final elongation 72°C - 5 min.

Next, the presence of amplification products was checked using horizontal electrophoresis in a 2% agarose gel. The luminescence of bands was recorded in all wells containing the PCR product, which indicates the presence of CP infection. The amplifier is purified on columns according to the manufacturer's instructions.

Next, a sequence reaction was performed according to the following protocol: deionized water - 5.5 μl; terminator - 0.5 µl; 5x buffer - 1 µl; primer - 1 µl forward or reverse (SEQ ID NO 1-6: SEQ ID NO 1: 5'-TAAATACCGACCTGCATGAATG-3', SEQ ID NO 2: 5'-CTCCATAAGAGCCAAAGCCC-3', SEQ ID NO 3: 5'- GGAAACCCTGAAGC AGC AACG-3', SEQ ID NO 4: 5'-CGCCTTCGC AATGAGT ATTCCT-3', SEQ ID NO 5: 5'-ATC ATAGGGCGTGCTTTACC-3', SEQ ID NO 6: 5'-AAAGGTT AGGC AG ACGGCTT-3 ', Appendix 1); PNR product - 2 µl. Sequence reaction mode: denaturation 96°C - 1 min.; 25 cycles: denaturation 96°C - 10 sec.; primer annealing 60°C - 5 sec.; elongation - 60°C - 4 min.

Next, the amplifier was purified using the sorbent method. Subsequent separation of nucleotides is carried out by capillary gel electrophoresis on a genetic analyzer. To determine nucleotide substitutions at target points, the obtained sequences are aligned with the reference sequence. Alignment of the sequences of the DNA sample of patient No. 3 with reference sequences is presented in Fig. 5.

As a result of the study, the patient was diagnosed with CP infection - S16+, resistance to clarithromycin was found - S23 (2142A, 2143G) - (mut), resistance to levofloxocin was not detected - GyrA 87 codon (259A, 261C) - wild type (wt); GyrA 91 codon (271G, 272A) - wild type (wt) (Fig. 6.).

According to the proposed method, a study was conducted on the presence of CP and its resistance to clarithromycin and levofloxacin in 16 patients. The sensitivity of CP to the antibiotics under study, established by this method, made it possible to carry out successful eradication therapy.

Information sources:

1. Oleastro M., et al. Real-Time PCR Assay for Rapid and Accurate Detection of Point Mutations Conferring Resistance to Clarithromycin in Helicobacter pylori // Journal of clinical microbiology, Vol. 41, No. 1. - 2003, p. 397-4025.

2. patent CN 200410073454, published 2007-01-17 “Chip for detecting polymorphism of the Helicobacter pylori clarithromycin resistance gene.”

3. Stepanov Yu.M., Budzak I.Ya. Maastricht Consensus - 5: analytical review of provisions // Gastroenterology, v. 51, no. 1. - 2017., pp. 36-45.

4. Andreev D.N., Maev I.V., Kucheryavyi Yu.A. Resistance of Helicobacter pylori in the Russian Federation: meta-analysis of studies over the past 10 years // Therapeutic archive, 11, 2020. - pp. 24-30. Doi: 10.2644/00403660.2020.11.000795.

5. Savoldi A., et al. Prevalence of Antibiotic Resistance in Helicobacter pylori: A Systematic Review and Meta-analysis in World Health Organization Regions // Gastroenterology. - 2018. 155(5): 1372-1382.

6. Bordin D.S. and others. “Methods for diagnosing Helicobacter pylori infection.” Guidelines. - Moscow, 2019.

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List of nucleotide sequences (primers)

<110> GBUZ MKSC im. A.S. Loginova

<120> Method for diagnosing Helicobacter pylori infection and its resistance to

clarithromycin and levofloxacin

<160> SEQ ID NO 1 primer forward

<210> SEQ ID NO 1

<211> 22

<212> DNA

<213> Helicobacter pylori

<400>Sequence

taaataccgacctgcatgaatg

<160> SEQ ID NO 2 primer reverse

<210> SEQ ID NO 2

<211> 20

<212> DNA

<213> Helicobacter pylori

<400>Sequence

ctccataagagccaaagccc

<160> SEQ ID NO 3 primer forward

<210> SEQ ID NO 3

<211> 21

<212> DNA

<213> Helicobacter pylori

<400>Sequence

ggaaaccctgaagcagcaacg

<160> SEQ ID NO 4 primer reverse

<210> SEQ ID NO 4

<211> 22

<212> DNA

<213> Helicobacter pylori

<400>Sequence

cgccttcgcaatgagtattcct

<160> SEQ ID NO 5 primer forward

<210> SEQ ID NO 5

<211> 20

<212> DNA

<213> Helicobacter pylori

<400>Sequence

atcatagggcgtgctttacc

<160> SEQ ID NO 6 primer reverse

<210> SEQ ID NO 6

<211> 20

<212> DNA

<213> Helicobacter pylori

<400>Sequence

aaaggttaggcagacggctt

List of reference nucleotide sequences of genes S23, GyrA, S16

<110> GBUZ MKSC im. A.S. Loginova

<120> Method for diagnosing Helicobacter pylori infection and its resistance to

clarithromycin and levofloxacin

<160> SEQ ID NO ref S23

<210> SEQ ID NO 1

<211> 264

<212> DNA

<213> Helicobacter pylori

<400> Sequence 1

Aggttaagaggatgcgtcagtc gcaagatgaagcgttgaattgaagcccgagtaaacggc 60

ggccgtaactataacggtcctaaggtagcgaaattccttgtcggttaaataccgaccatg 120

aatggcgtaacgagatgggagctgtctcaaccagagattcagtgaaattgtagtggaggt 180

gaaaattcctcctacccgcggcaagacgg aa agaccccgtggacctttactacaacttag 240

cactgctaatgggaatatcatgcg 300

<160> SEQ ID NO ref GyrA

<210> SEQ ID NO 2

<211> 415

<212> DNA

<213> Helicobacter pylori

<400> Sequence 2

atcatagggcgtgctttaccggacgctagagatggcttaaagcccgtgcataggcgtatt 60

ttgtatgcgatgcatgaattaggcctcacttccaaagtcgcttataaaaaaagcgctagg 120

attgtgggtgatgtgattggtaaataccacccccatggcgataacgcggtttatgatgcg 180

ctagtgagaatggcgcaagatttttctatgcgtttggaattagtggatgggcaaggcaac 240

tttggctctattgatggcgataacgctgcagcgatgcgttacactgaagccagaatgacc 300

aaggcgagtgaagaaattttaagagatattgataaagacaccattgattttgtgcctaat 360

tacgatgacaccttaaaagagcccgatattttaccaagccgtctgcctaaccttt 520

<160> SEQ ID NO ref S16

<210> SEQ ID NO 3

<211> 330

<212> DNA

<213> Helicobacter pylori

<400> Sequence 3

ggaaaccctgaagcagcaacgccgcgtggaggatgaaggttttaggattgtaaactcctt 60

ttgttagagaagataatgacggtatctaacgaataagcaccggctaactccgtgccagca 120

gccgcggtaatacggagggtgcaagcgttactcggaatcactgggcgtaaagagcgcgta 180

ggcgggatagtcagtcaggtgtgaaatcctatggcttaaccatagaactgcatttgaaac 240

tactattctagagtgtgggagaggtaggtggaattcttggtgtaggggtaaaatccgtag 300

agatcaagaggaatactcattgcgaaggcg 360

<---

Claims (1)

A method for detecting Helicobacter pylori infection and its resistance to clarithromycin and levofloxacin in a biopsy sample of the patient's gastric mucosa, including performing a polymerase chain reaction using a PCR reaction solution and primers SEQ ID NO 1: 5'-TAAATACCGACCTGCATGAATG-3', SEQ ID NO 2 : 5'-CTCCATAAGAGCCAAAGCCC-3', SEQ ID NO 3: 5'-GGAAACCCTGAAGCAGCAACG-3', SEQ ID NO 4: 5'-CGCCTTCGCAATGAGTATTCCT-3', SEQ ID NO 5: 5'-ATCATAGGGCGTGCTTTACC-3', SEQ ID NO 6: 5'-AAAGGTTAGGCAGACGGCTT-3', purification of the PCR product, sequence reaction, purification of the product of the sequence reaction, detection and analysis of the result, while the test system contains a set of reagents for PCR, buffer, MgCl2, dNTP, Taq polymerase , DNA sample and primer set, SEQ ID NO 1: 5'-TAAATACCGACCTGCATGAATG-3', SEQ ID NO 2: 5'-CTCCATAAGAGCCAAAGCCC-3', SEQ ID NO 3: 5'-GGAAACCCTGAAGCAGC AACG-3', SEQ ID NO 4: 5'-CGCCTTCGCAATGAGTATTCCT-3', SEQ ID NO 5: 5'-ATCATAGGGCGTGCTTTACC-3', SEQ ID NO 6: 5'-AAAGGTTAGGCAGACGGCTT-3'.