RU2839157C1 - Sets of oligonucleotides for detecting dna of helicobacter pylori bacterium in clinical material by real-time polymerase chain reaction - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to a set of oligodeoxyribonucleotide primers. Said kit is intended for identifying DNA of the bacterium Helicobacter pylori in a clinical material by real-time polymerase chain reaction and contains a forward primer with the sequence SEQ ID NO: 1, reverse primer with sequence SEQ ID NO: 2 and a fluorescent-labelled DNA probe with the sequence SEQ ID NO: 3.

EFFECT: present invention provides a set of oligonucleotides, which enables to efficiently detect Helicobacter pylori DNA.

1 cl, 3 ex

Description

The invention relates to the field of medicine, in particular to molecular genetic diagnostics in the field of gastroenterology. Three sets of oligodeoxyribonucleotide primers and fluorescently labeled DNA probes are proposed, each of which can be independently used to identify DNA of the Helicobacter pylori bacterium in clinical material using the polymerase chain reaction in real time (PCR-RT). The invention can be used in clinical laboratory diagnostics and for scientific research purposes.

Helicobacter pylori is a gram-negative spiral-shaped microaerophilic flagellated bacterium capable of adapting to extremely acidic conditions of the gastric environment, causing chronic active gastritis in all infected individuals [Camilo V., Sugiyama T., Touati E. Pathogenesis of Helicobacter pylori infection // Helicobacter. 2017. Vol. 22. Suppl. 1. DOI: https://doi.org/10.1111/hel.12405 ]. Helicobacter pylori is transmitted from person to person, most often in childhood, mainly by the fecal-oral route [Bordin D.S., Shengelia M.I., Ivanova V.A., Voynovan I.N. Helicobacter pylori , clinical significance and principles of diagnosis // Infectious diseases: News. Opinions. Training. 2022. No. 1 (40). DOI: https://doi.org/10.33029/2305-3496-2022-11-1-119-129 ].

Helicobacter pylori is known to colonize the gastric mucosa (GM) and in many infected individuals causes chronic gastritis, which can progress to the development of gastric ulcer and duodenal ulcer, atrophic gastritis, MALT lymphoma (MALT – mucosa-associated lymphoid tissue; lymphoma from mucosa-associated lymphoid tissue) and gastric adenocarcinoma [FitzGerald R., Smith SM An Overview of Helicobacter pylori Infection // Methods in Molecular Biology. 2021. Vol. 2283. P. 1–14. DOI: https://doi.org/10.1007/978-1-0716-1302-3_1 ]. There is evidence linking Helicobacter pylori with the development of essential iron deficiency anemia, idiopathic thrombocytopenic purpura and vitamin B12 deficiency [Sheptulin A.A. Main provisions of the Maastricht-VI consensus meeting (2022) on the diagnosis and treatment of Helicobacter pylori infection // Russian Journal of Gastroenterology, Hepatology, Proctology. 2022. No. 32 (5). P. 70–74. DOI: https://doi.org/10.22416/1382-4376-2022-32-5-70-74 ]. Prevention of these diseases is based on early detection and treatment of Helicobacter pylori infection.

Effective diagnostics of Helicobacter pylori requires a combination of various methods, such as histological analysis of gastric mucosa and duodenal biopsies, bacteriological method, C-urea breath test and stool analysis for Helicobacter pylori antigen. Also, a very widespread method for detecting Helicobacter pylori is the use of molecular genetic methods, including RT-PCR. The use of RT-PCR in combination with classical diagnostic methods can significantly reduce the number of false positive results. The method demonstrates sensitivity and specificity of up to 95% [Szymczak A., Ferenc S., Majewska J., Miernikiewicz P. et al. Application of 16S rRNA gene sequencing in Helicobacter pylori detection // PeerJ. 2020. Vol. 8. P. e9099. DOI: https://doi.org/10.7717/peerj.9099 ].

The closest analogues in Russia are the reagent kits for detection of Helicobacter pylori DNA by the polymerase chain reaction method "Helicobacter pylori", manufactured by DNA-Technology TS LLC and "RealBest Helicobacter pylori DNA", manufactured by Vector-Best JSC. However, the list of compatible recording amplifiers for the operation of these kits includes only devices manufactured by Bio-Rad (USA), NPO DNA-Technology LLC (Russia) and Qiagen (Germany).

A method for detecting Helicobacter pylori DNA is known, described in patent KR100846494B1, however, the cagA gene region (not 16S rRNA and 23S rRNA) is used for detection, and gel electrophoresis is used for detection of amplicons instead of fluorescent probes, thereby increasing the likelihood of contamination with amplicons, which can lead to false positive results.

The technical result is the creation of sets of oligonucleotides that can be independently used to detect Helicobacter pylori DNA.

The aim of the present invention is to develop a set of unique highly specific oligodeoxyribonucleotide primers for detecting the bacterium Helicobacter pylori by PCR with hybridization-fluorescence detection in real time in clinical material, taking into account the possibility of using an extended list of compatible amplifiers.

The task is solved by selecting oligodeoxyribonucleotide primers and a fluorescently labeled DNA probe to identify the DNA of the Helicobacter pylori bacterium using the polymerase chain reaction method with detection of amplification products in real time. A total of 3 kits were selected that provide similar analytical and diagnostic characteristics in RT-PCR, with the following structures in the first kit: forward primer HP_F_N1 (SEQ ID NO 4): 5'-CGGGATGCTTAATGCGTTAGC-3', reverse primer HP_R_N1 (SEQ ID NO 5): 5'-GCTGGAACATTACTGACGCTGATT-3', fluorescently labeled DNA probe HP_P_N1 (SEQ ID NO 6): 5'-FAM-ACTAAGCCCTCCAACAACTAGCATCCATC-BHQ1-3'; for the second set: forward primer HP_23S_F1n(60) (SEQ ID NO 7): 5`-GAAGGTTAAGAGGATGCGTC-3`, reverse primer HP_23S_R3(60.9) (SEQ ID NO 8): 5`-AATCTCTGGTTGAGACAGCT-3`, fluorescently labeled DNA probe HP_23S_P2-1 (SEQ ID NO 3): 5`-FAM-TTGAAGCCCGAGTAAACGGCGGC-BHQ1-3'; for the third set: forward primer HP_23S_F2-5 (SEQ ID NO 1): 5`-TCAGTCGCAAGATGAAGCGT-3`, reverse primer HP_23S_R3 (SEQ ID NO 2): 5`-GAATCTCTGGTTGAGACAGCTCC-3`, fluorescently labeled DNA probe HP_23S_P2-1 (SEQ ID NO 3): 5`-FAM-TTGAAGCCCGAGTAAACGGCGGC-BHQ1-3'.

Each of the oligonucleotide sets can be used independently to detect Helicobacter pylori DNA by PCR.

To use each of the oligonucleotide sets, it is necessary to prepare a PCR mixture (total volume – 25 µl), including:

forward primer at a concentration of 0.4 μM,

reverse primer at a concentration of 0.4 μM,

probe at a concentration of 0.2 μM,

PCR buffer containing salt solutions, thermostable hot-start DNA polymerase, deoxynucleotide triphosphates (dNTPs);

DNA matrix (15 µl).

To control the reaction progress, a positive control sample (PCS) and a negative control sample (NCS) are used in the setup. PCS is a solution of plasmid DNA with a synthetic insert of amplified DNA fragments: specific fragments of the Helicobacter pylori bacterium genome. NCS is deionized water free of DNases. NCS undergoes the stage of NC isolation with the addition of VCS.

This set is used as follows.

1. Carry out DNA extraction from biological material (coolant, feces, saliva) using specialized DNA extraction kits (sample preparation reagent kits, not the subject of this patent); during this procedure, DNA is extracted from the biological material, which in turn is used for PCR;

2. Prepare the required number of 0.1–0.2 ml test tubes for PCR (the number of test tubes is determined based on the number of samples being tested + 1 test tube for OKO + 1 test tube for PKO);

3. In a separate disposable sterile Eppendorf tube with a volume of 1.5-2.0 ml, prepare a reaction mixture consisting of oligonucleotides and PCR buffer;

4. Add 10 µl of the prepared reaction mixture to each PCR tube;

5. Add 15 µl of the extracted DNA to the appropriate test tubes for the samples being studied. The DNA preparation is not added to the test tubes with the PCO and OCO;

6. Add PKO and OKO into the appropriate test tubes;

7. Mix the contents of the test tubes, then remove the drops from the walls by briefly centrifuging for 1–3 seconds in a microcentrifuge-vortex;

8. Place the tubes in the reaction module of the real-time PCR device. Amplification is carried out according to the following program: 1 cycle: 95°C for 2 minutes; 5 cycles: 95°C for 15 seconds, 64°C for 20 seconds; 40 cycles: 95°C for 15 minutes, 64°C for 20 seconds (at this stage, detection of the fluorescent signal via the FAM/Green channel is required). The following devices can be used for PCR: CFX96 (BioRad, USA), DTprime (NPO DNA-Technology LLC, Russia), Rotor-Gene Q (Qiagen, Germany), QuantStudio 5 (Thermo Fisher Scientific, USA), Gentier 96 (Tianlong, China).

After amplification and detection of amplification products in real time, the results are interpreted. The result is considered positive, i.e. Helicobacter pylori DNA is detected in the sample under study, with an increase in the fluorescence level in the corresponding tubes; in this case, the fluorescence growth curves should have an S-shape and also intersect with the threshold line before the 40th amplification cycle.

It is recommended to perform DNA extraction on the day of clinical material collection. Biopsy specimens may be transported and stored at a temperature of 2°C to 8°C for no more than 24 hours. If it is impossible to deliver the material to the laboratory within 24 hours, a single freezing of the material is allowed.

Pre-treatment of feces samples is required. When examining native feces without prior freezing, prepare a fecal suspension (if the feces have a watery consistency, do not prepare a suspension) from 0.8 ml of phosphate buffer (or sterile isotonic sodium chloride solution) and 0.1 g (0.1 ml) of feces. If it is impossible to examine the material within 24 hours and/or long-term storage is required, glycerol is added to a 10–20% suspension of feces in phosphate buffer (or sterile isotonic sodium chloride solution) at a final concentration of 10–15%. Samples prepared in this way are frozen only after thorough homogenization and exposure to glycerol for 30–40 minutes.

It should be understood that a person skilled in the art can, without further experimentation and taking into account the present description, apply the present invention in practice to its full extent. Therefore, the description of the invention given in this context is presented for illustration, without reducing the scope of the claims.

Clinical example #1

A 27-year-old patient G.A.N. was admitted to the gastroenterology department of the KFT Clinics of the Federal State Budgetary Educational Institution of Higher Education "Samara State Medical University" of the Ministry of Health of the Russian Federation with complaints of pain in the epigastrium and nausea. During esophagogastroduodenoscopy, multiple fresh erosions were found in the antral part of the stomach, the mucous membrane was edematous, and biopsy material was taken. As a result of endoscopic and cytological studies, the diagnosis of pyloric helicobacteriosis was confirmed. The biopsy material was examined histologically (average Helicobacter pylori contamination). Feces were also collected from patient G.A.N.

From the biopsy material after treatment with proteinase K, nucleic acids were extracted using the reagent kit for isolating nucleic acids from biological material NK-sorbent for in vitro diagnostics in the Tissue version (OOO NPF Litekh, Russia). From the fecal samples after sample preparation (preparation of a fecal suspension from 0.8 ml of phosphate buffer (or sterile isotonic sodium chloride solution) and 0.1 g (0.1 ml) of feces), nucleic acids were extracted using the PROBA-NK reagent kit for isolating nucleic acids (OOO NPO DNA-Technology, Russia). The obtained DNA samples in a volume of 15 μl were added to microtubes with reaction mixtures prepared in them for the first set of primers in a volume of 10 μl, a polymerase chain reaction was carried out with detection of amplification products in real time on a DTprime amplifier (NPO DNA-Technology LLC, Russia).

The first set of primers consisted of: forward primer HP_F_N1 (SEQ ID NO 4): 5'-CGGGATGCTTAATGCGTTAGC-3', reverse primer HP_R_N1 (SEQ ID NO 5): 5'-GCTGGAACATTACTGACGCTGATT-3', fluorescently labeled DNA probe HP_P_N1 (SEQ ID NO 6): 5'-FAM-ACTAAGCCCTCCAACAACTAGCATCCATC-BHQ1-3'.

Amplification was carried out according to the program: 1 cycle: 95°C 2 minutes; 5 cycles: 95°C 15 seconds, 64°C 20 seconds; 40 cycles: 95°C 15 minutes, 64°C 20 seconds (at this stage, detection of the fluorescent signal via the FAM/Green channel is required). When studying the results, an increase in the fluorescence level was recorded in the corresponding tubes, while the fluorescence growth curves had an S-shape, intersecting with the threshold line at the 25th amplification cycle. Therefore, Helicobacter pylori DNA was detected in the samples, which confirms the diagnosis.

Clinical example #2

A 35-year-old patient Ch.A.V. was admitted to the gastroenterology department of the KFT Clinics of the Federal State Budgetary Educational Institution of Higher Education "Samara State Medical University" of the Ministry of Health of the Russian Federation with complaints of pain in the epigastrium and a single episode of vomiting. During esophagogastroduodenoscopy, fresh multiple erosions were found in the antral part of the stomach, the mucous membrane was edematous, slightly hyperemic, and biopsy material was taken. As a result of endoscopic and cytological studies, the diagnosis of pyloric helicobacteriosis was confirmed. The biopsy material was examined histologically (low Helicobacter pylori contamination) . Feces were also collected from patient Ch.A.V.

From the biopsy material after treatment with proteinase K, nucleic acids were extracted using a reagent kit for the extraction of nucleic acids from biological material NK-sorbent for in vitro diagnostics in the Tissue version (OOO NPF Litekh, Russia). From fecal samples after sample preparation (preparation of a fecal suspension from 0.8 ml of phosphate buffer (or sterile isotonic sodium chloride solution) and 0.1 g (0.1 ml) of feces), nucleic acids were extracted using a reagent kit for the extraction of nucleic acids PROBA-NK (OOO NPO DNA-Technology, Russia). The obtained DNA samples in a volume of 15 μl were added to microtubes with reaction mixtures prepared in them for the second set of primers in a volume of 10 μl, and a polymerase chain reaction was carried out with detection of amplification products in real time on a DTprime amplifier (NPO DNA-Technology LLC, Russia).

The second set of primers consisted of: forward primer HP_23S_F1n(60) (SEQ ID NO 7): 5`-GAAGGTTAAGAGGATGCGTC-3`, reverse primer HP_23S_R3(60.9) (SEQ ID NO 8): 5`-AATCTCTGGTTGAGACAGCT-3`, fluorescently labeled DNA probe HP_23S_P2-1 (SEQ ID NO 3): 5`-FAM-TTGAAGCCCGAGTAAACGGCGGC-BHQ1-3'.

Amplification was carried out according to the program: 1 cycle: 95°C 2 minutes; 5 cycles: 95°C 15 seconds, 64°C 20 seconds; 40 cycles: 95°C 15 minutes, 64°C 20 seconds (at this stage, detection of the fluorescent signal via the FAM/Green channel is required). When studying the results, an increase in the fluorescence level was recorded in the corresponding tubes, while the fluorescence growth curves had an S-shape, intersecting with the threshold line at the 27th amplification cycle. Therefore, Helicobacter pylori DNA was detected in the samples, which confirms the diagnosis.

Clinical example #3

Patient Sh.V.R., aged 56, was admitted to the gastroenterology department of the KFT Clinics of the Federal State Budgetary Educational Institution of Higher Education “Samara State Medical University” of the Ministry of Health of the Russian Federation with complaints of heartburn, a feeling of “heaviness” and pain in the epigastrium. During esophagogastroduodenoscopy, pronounced erosions in the antral part of the stomach were detected, the mucous membrane was edematous, diffusely hyperemic with areas of dystrophy, biopsy material was taken. As a result of endoscopic and cytological studies, the diagnosis of pyloric helicobacteriosis was confirmed. The biopsy material was examined histologically (high colonization of Helicobacter pylori) . Feces were also collected from patient Sh.V.R.

From the biopsy material after treatment with proteinase K, nucleic acids were extracted using a reagent kit for the extraction of nucleic acids from biological material NK-sorbent for in vitro diagnostics in the Tissue version (OOO NPF Litekh, Russia). From fecal samples after sample preparation (preparation of a fecal suspension from 0.8 ml of phosphate buffer (or sterile isotonic sodium chloride solution) and 0.1 g (0.1 ml) of feces), nucleic acids were extracted using a reagent kit for the extraction of nucleic acids PROBA-NK (OOO NPO DNA-Technology, Russia). The obtained DNA samples in a volume of 15 μl were added to microtubes with reaction mixtures prepared in them for the third set of primers in a volume of 10 μl, a polymerase chain reaction with detection of amplification products in real time was carried out on a DTprime amplifier (NPO DNA-Technology LLC, Russia).

The third set of primers consisted of: forward primer HP_23S_F2-5 (SEQ ID NO 1): 5`-TCAGTCGCAAGATGAAGCGT-3`, reverse primer HP_23S_R3 (SEQ ID NO 2): 5`-GAATCTCTGGTTGAGACAGCTCC-3`, fluorescently labeled DNA probe HP_23S_P2-1 (SEQ ID NO 3): 5`-FAM-TTGAAGCCCGAGTAAACGGCGGC-BHQ1-3'.

Amplification was carried out according to the program: 1 cycle: 95°C 2 minutes; 5 cycles: 95°C 15 seconds, 64°C 20 seconds; 40 cycles: 95°C 15 minutes, 64°C 20 seconds (at this stage, detection of the fluorescent signal via the FAM/Green channel is required). When studying the results, an increase in the fluorescence level was recorded in the corresponding tubes, while the fluorescence growth curves had an S-shape, intersecting with the threshold line at the 23rd amplification cycle. Therefore, Helicobacter pylori DNA was detected in the samples, which confirms the diagnosis.

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Claims (4)

A set of oligodeoxyribonucleotide primers for the identification of Helicobacter pylori bacterium DNA in clinical material using real-time polymerase chain reaction, which contains: forward primer HP_23S_F2-5 (SEQ ID NO: 1): 5`-TCAGTCGCAAGATGAAGCGT-3`, Reverse primer HP_23S_R3 (SEQ ID NO: 2): 5`-GAATCTCTGGTTGAGACAGCTCC-3`, fluorescently labeled DNA probe HP_23S_P2-1 (SEQ ID NO: 3): 5`-FAM-TTGAAGCCCGAGTAAACGGCGGC-BHQ1-3'.