RU2732923C1 - Method and kit for detecting campylobacter bacteria in patients with inflammatory intestinal diseases - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to biotechnology. According to the invention, formalin-fixed sections of biopsy materials fixed by formalin and paraffin-embedded biopsies of affected intestinal areas, obtained by colonoscopy in patients with nonspecific ulcerative colitis and Crohn's disease, using PCR with the genus specific primers homologous to the genus specific fragment of 16S rRNA bacteria of the genus Campylobacter: C. jejuni, C. coli and C. upsaliensis. Kit for detecting Campylobacter bacteria contains 6 components.

EFFECT: use of group of inventions simplifies and reduces duration of analysis.

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Description

The group of inventions relates to medicine, namely to medical microbiology and can be used to detect pathogenic for humans and animals (thermophilic) bacteria of the Campylobacter genus in intestinal tissue samples enclosed in paraffin (TPF) in patients with inflammatory bowel diseases (IBD).

Bacteria of the genus Campylobacter are currently considered as the main cause of bacterial gastroenteritis worldwide [Anonymous Monitoring the Incidence and Causes of Diseases Potentially Transmitted by Food in Australia: Annual Report of the OzFoodNet Network, 2010. Communicable Diseases Intelligence. 2012; 36: E213-E241; Eurosurveillance Editorial Team. The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-Borne Outbreaks in 2010. Euro Surveillance. 2012; 17:21]. The most etiologically significant among them are Campylobacter jejuni and Campylobacter coli [Skirrow M.B. Diseases Due to Campylobacter, Helicobacter and Related Bacteria. Journal of Comparative Pathology. 1994; 111: 113-149. http://dx.doi.org/10.1016/S0021-9975(05)80046-5]. The epidemiological significance of a number of other representatives of Campylobacter spp., Such as Campylobacter concisus, Campylobacter upsaliensis, Campylobacter hyointestinalis and Campylobacter fetus, which can also cause diseases in humans [Linton D., Owen R.J. and Stanley J. Rapid Identification by PCR of the Genus Campylobacter and of Five Campylobacter Species Enteropathogenic for Man and Animals. Research in Microbiology. 1996; 147: 707-718. http://dx.doi.org/10.1016/S0923-2508(97)85118-2; Kulkarni S.P., Lever S., Logan J.M., Lawson A.J., Stanley J., Shafi M.S. Detection of Campylobacter Species: A Comparison of Culture and Polymerase Chain Reaction Based Methods. Journal of Clinical Pathology. 2002; 55: 749-753; Sarnie A., Obi C.L., Barrett L.J., Powell S.M., Guerrant R.L. Prevalence of Campylobacter Species, Helicobacter pylori and Arcobacter Species in Stool Samples from the Venda Region, Limpopo, South Africa: Studies Using Molecular Diagnostic Methods. Journal of Infection. 2007; 54: 558-566].

Along with this, there are indications that Campylobacter spp. Are also important in the etiology of IBD, such as ulcerative colitis and / or Crohn's disease [Mahendran V., Riordan SM, Grimm MS, Tran TAT, Major J. et al. Prevalence of Campylobacter Species in Adult Crohn's Disease and the Preferential. Colonization Sites of Campylobacter Species in the Human Intestine. PLoS ONE. 2011; 6 (9): e25417. doi: 10.1371 / journal.pone.0025417]. In favor of the above is the fact that in the pathogenesis of inflammatory bowel diseases, the leading role of the autoimmune component of intestinal tissue damage, which can be initiated by Campylobacter bacteria, namely, their ability to penetrate the mucous membrane with its subsequent sensitization [Mahendran V., Riordan SM, Grimm M. C., Tran TAT, Major J. et al. Prevalence of Campylobacter Species in Adult Crohn's Disease and the Preferential. Colonization Sites of Campylobacter Species in the Human Intestine. PLoS ONE. 2011; 6 (9): e25417. doi: 10.1371 / journal.pone.0025417; Kaakoush N.O., Mitchell H.M. Campylobacter concisus - a new player in intestinal disease. Front. Cell. Infect. Microbiol. 03 February 2012 https://doi.org/10.3389/fcimb.2012.00004; Rubin D.C., Shaker A., Levin M.S. Chronic intestinal inflammation: inflammatory bowel disease and colitis-associated colon cancer. Front. Immunol., 08 May 2012 https://doi.org/10.3389/fimmu.2012.00107]. Accordingly, the autoimmune effects of Campylobacter spp. Can be prevented and / or stopped by the timely conduct of etiotropic antibiotic therapy due to the early detection and subsequent eradication of these microorganisms. However, this requires their reliable detection directly in the tissues of the affected areas of the intestine, in which Campylobacter spp. Can persist for a long time even if they are absent or not detected in feces.

Known methods for the detection and identification of Campylobacter spp. when using microscopy or culture [Methodical Guidelines (MP) 01 / 15702-8-34. Microbiological diagnosis of campylobacteriosis. 2008; Sanitary Rules (SP) 3.1.7.2816-10. Prevention of campylobacteriosis among humans. 2010]. However, microscopic methods are characterized by extremely low sensitivity and specificity. Culturing methods that involve the isolation and identification of pure cultures of Campylobacter spp. Are predominantly focused on the study of native faeces, which must be delivered to the laboratory in special transport media (Cary-Blair medium, Amies medium). Seeding is carried out after preliminary enrichment of the test material by the method of "membrane" filters [Boitsov A.G., Porin A.A. Device for the isolation of Campylobacter spp. From a sample. USSR author's certificate, No. 01576558 dated 07.07.1990], on selective media containing antibiotics in various combinations Skirrow M. (vancomycin + polymyxin B + trimethoprim), Blaser M. (vancomycin + polymyxin B + trimethoprim + cephalothin + amphotericin), Karmali M. (cefoperazone + vancomycin + cycloheximide), Matveeva Z. (rifampicin + polymyxin B + ristomycin + amphotericin), Minaev V. (rifampicin + fuzidin + cephalothin + amphotericin), etc.). For the cultivation of Campylobacter spp. Complex blood culture media have been proposed, including growth factors, selective additives (antibiotics) and chromogenic substrates, and therefore expensive and with limited shelf life, for example, Campy Food ID (Biomerieux, France) and Brilliance CampyCount (Oxoid, USA) media. Regardless of the nutrient media used, crops are incubated in a special gas-air medium for microaerophilic bacteria (5-7% O ₂ , 10% CO ₂ , 83-85% N ₂ or 6% O ₂ , 7% CO ₂ , 7% H ₂ , 80% N ₂ ), in CO ₂ incubators or anaerostats. Modifications of the culture method for the quantitative determination of bacteria of the genus Campylobacter in food products have been proposed [Guidelines (MUK 4.2.2321-08) Methods for the determination of bacteria of the genus Campylobacter in food]. The above methods are distinguished by extremely high material and labor intensity, the duration of the study varies from 3 to 7 days. Moreover, none of these methods can be used to detect bacteria of the genus Campylobacter in biopsies enclosed in paraffin, since this is necessarily preceded by fixation of tissue in formalin and the death of viable bacteria. This completely excludes the possibility of subsequent cultivation of Campylobacter spp. and their isolation in pure culture [Kirk KF, Nielsen HL, Thorlacius-Ussing O., Nielsen H. Optimized cultivation of Campylobacter concisus from gut mucosal biopsies in inflammatory bowel disease. Gut Pathog. 2016; 8: 27. DOI 10.1186 / sl3099-016-0111-7].

Agglutination tests (Microscreen (Lucron), INDX-Campy (Biotrading), Dryspot Campylobacter Test (Oxoid), BBL-Campyslide (BD)) and immunochromatographic tests can be classified as express tests for the detection of Campylobacter spp., Which do not involve the cultivation of the desired microorganisms. [MP 24 ФЦ / 976-2004. Methods for detecting pathogenic microorganisms using immunochromatographic express tests manufactured by Merck (Germany)]. However, they can only be used for testing serum or native faeces, not paraffin-embedded tissue samples.

A known method for determining the infection of the stomach Campylobacter pyloridis by measuring the concentration of urea in the basal and stimulated by histamine or pentagastrin fractions of gastric juice before and after antibacterial therapy [Copyright certificate SU 1659850 A1, IPC G01N 33/48, BI N 24, 1991], but it has low specificity, sensitivity and is also inapplicable for TPF.

Known set of reagents for the detection of DNA Campylobacter spp. in food by polymerase chain reaction (PCR) with hybridization-fluorescence detection "AmpliSens® Campylobacter spp.-FL" by PCR [https: //www.interlabservice: ru / upload / iblock / dc9 / Campvlobacter% 20spp.-FL% 20060219.pdf]. However, the method is designed to study DNA samples isolated from liquid selective nutrient media for the cultivation of bacteria of the genus Campylobacter for the purpose of primary enrichment of the test material and the isolation of these microorganisms from food. This excludes the possibility of research by this method using the proposed set of reagents fixed in formalin and embedded in paraffin tissue samples.

The closest analogue of the method for detecting bacteria of the genus Campylobacter is a method for determining in a biological sample of DNA pathogenic microorganisms causing acute intestinal infections, including Campylobacter jejuni, which includes isolating DNA from a biological sample containing a pathogen, obtaining forward and reverse primers specific to DNA of one or more OKI markers and microchip, carrying out PCR in the presence of a DNA sample and primers specific to the specified one or more markers, applying PCR products to the working area of the biochip, incubation under hybridization conditions, detection of the formed hybridization complexes on a biochip that contains differentiating oligonucleotides of a certain sequence [patent RU 2509804, 2014]. The disadvantages of the prototype are the duration, high cost and labor intensity of the method.

The closest analogue of the kit for identifying bacteria of the genus Campylobacter is a kit for detecting DNA of pathogenic microorganisms causing acute intestinal infections, including Campylobacter jejuni, comprising a matrix containing amino groups with a density of 1-2 groups / nm ² ; a reaction mixture containing 1 μM oligonucleotide and 60 mM 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride in 0.1 M 1-methylimidazole, pH 7.0; washing solutions - 0.5 mM NaCl containing 0.1% sodium dodecyl sulfate and distilled water; oligonucleotides modified with a phosphate group (p) at the 5'-position of SEQ ID NOs: 59, 68, 90 and 91; for lysis - physiological solution, lysis buffer in deionized water (Tris-HCl, pH 6.8 - 0.05 M, guanidine thiocyanate - 3.13 M, GTC, ethylenediamine tetraacetate (EDTA) - 0.025 M, sarcosyl - 1, buffer 1 ( Tris-HCl, pH 6.8 - 0.0585 M, guanidine hydrochloride - 2.34 M, and EDTA - 0.0243 M), buffer 2 (1 M Tris-HCl, pH 8.0 with a final concentration of 0.05 M ); elution buffer (Chelex 100 - 5%, Tris-HCl, pH 8.0 - 0.05 M); PCR mixture - compositions of oligonucleotide primers SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, and 1 μl of Taq DNA polymerase, 2x reaction buffer is prepared in deionized water (1 M Tris-HCl, pH 8.8-0.16 M; (NH ₄ ) ₂ SO ₄ - 0.032 M; MgCl ₂ - 0.004 M; dNTP mixture - 0.004 M; Tween 20 - to a final concentration of 0.04%); PC-positive control sample - a mixture of recombinant plasmids obtained on the basis of pUC18 and containing the sequence of marker genes of each pathogen at a concentration of 5 pg / μl (or 1.5 × 10 ⁶ DNA molecules in 1 μl); negative control sample - recom binant plasmid obtained on the basis of pUC18 and containing the sequence of the GE13 marker at a concentration of 50 pg / μl (or 15 × 10 ⁶ DNA molecules in 1 μl). After the end of the amplification program, a solution of exonuclease in a buffer solution at a concentration of 2 units. activity / μl; stop solution - EDTA on deionized water at a concentration of 0.020 M. For hybridization of PCR products on biochips, a mixture (2.5 μl 20 × SSC, 2 μl 1% sodium dodecyl sulfate); solutions for chips: 2 × SSC, 0.1% SDS - 1 time 5 minutes; 1 × SSC, 0.1% sodium dodecyl sulfate - once 5 minutes; 0.5 × SSC - 2 times 5 minutes each [patent RU 2509804, 2014]. The kit provides the detection of 10-10 ³ DNA copies, along with a number of pathogens of only one campylobacter species - Campylobacter jejuni.

The objective of the present group of inventions is to develop a highly sensitive and specific method and kit for accelerated laboratory detection of pathogenic representatives of Campylobacter bacteria in paraffin-embedded intestinal tissue samples in patients with inflammatory bowel diseases.

The technical result achieved by using a group of inventions is to simplify and reduce the duration of the study.

The essence of the proposed group of inventions is as follows.

During colonoscopy, a biopsy specimen is taken from the affected area of the intestine, the biopsy specimen is fixed in formalin and embedded in paraffin in the conventional way [G.A. Merkulov. Course of pathological and histological techniques. - 5th ed., Rev. and add. Leningrad: Medicine. Leningrad. department, 1969.- 423 p.]. For the isolation of Campylobacter spp. from intestinal tissue samples, embedded in paraffin, sections with a thickness of 1-2 mm (4-6 sections per sample) are made, placed in eppendorf tubes, which are incubated in a portable thermostat at 93 ° C to melt the paraffin. Without removing the tubes from the thermostat, a component of 1-10 µl of magnetic particles ("Sileks" MagNA, Russia) is quickly added to each sample with the analyzed sample and suspended on a vortex. Place the tubes in a portable thermostat (93 ° C) and, without removing, add 100 μl of component 2 to each - binding buffer for lysis, containing 4M guanidine thiocyanate, 100 mM Tris-HCl buffer (pH 7.4), 2 mM ethylenediamine tetraacetate (EDTA) (pH 8.0), 2% Triton X-100. The tubes are then resuspended on a vortex and placed back in a portable oven (93 ° C). Then each of the test tubes is placed in turn in a magnetic rack. Using a dispenser, the light phase (residues of paraffin and tissue detritus) is taken from the test tubes and discarded, without touching the sediment from the magnetic particles. Then, 200 μl of component 3 is added to each sample - a washing solution containing 70% Ethanol, 20 mM NaCl, 10 mM Tris-HCl (pH 7.4), 2 mM EDTA (pH 8.0), vortexed for 10-15 seconds ... Place the test tubes in a magnetic rack, then select and discard the transparent phase, without touching the sediment from the magnetic particles. Tubes with open lids are dried at 65 ° C for 5-10 minutes. In each tube, add 50 μl of component 4 - elution buffer containing 10 mM Tris-HCl (pH 7.4), 1 mM EDTA (pH 8.0), stir for 5-10 sec, and precipitate magnetic particles in a magnetic stand. The supernatant containing the purified DNA is taken into clean labeled tubes. PCR is performed.

To do this, mix the isolated DNA (1 μl / sample) and the detection mixture (25 μl / sample), including 2.5 μl Taq buffer, 2.5 μl DNTP, 16.5 μl deionized water, 0.5 μl Taq polymerase , 1 μl each of primers CampF (5'-AGAACACGCGGACCTATATA-3 ') and CampR (5'-CGATGCATCCAGGTAATGTAT-3') - genus-specific primers homologous to the genus-specific fragment of 16S rRNA of bacteria of the genus Campylobacterlobacteri On the surface of each reaction mixture, 50 μl of mineral oil is layered. Amplification mode: denaturation (95 ° C, 1 min), then - 30 amplification cycles (DNA denaturation (95 ° C, 30 sec), annealing (60 ° C, 30 sec), elongation (72 ° C, 30 sec).

Then, the amplification products are detected by horizontal electrophoresis in agarose gel by comparing the electrophoretic mobility of the obtained amplicons with the mobility of the DNA Ladder Mix molecular weight marker (Fermentas, Lithuania). In the presence of an amplification product corresponding to a size of 256 nucleotide pairs (bp), it is concluded that the investigated tissue samples contain the main pathogenic representatives of Campylobacter spp. (Campylobacter jejuni, Campylobacter coli and Campylobacter upsaliensis).

Kit for PCR detection of Campylobacter spp. in tissue samples, embedded in paraffin, contains component 1: 10 µl of a suspension of magnetic particles ("Sileks" MagNA, Russia)); component 2: binding buffer for lysis containing 4M guanidine thiocyanate, 100 mM Tris-HCl buffer (pH 7.4), 2 mM EDTA (pH 8.0), 2% Triton X-100; component 3: wash solution containing 70% Ethanol, 20 mM NaCl, 10 mM Tris-HCl (pH 7.4), 2 mM EDTA (pH 8.0), component 4: elution buffer containing 10 mM Tris-HCl (pH 7.4) , 1 mM EDTA (pH 8.0); component 5: detection mixture containing 2.5 μl Taq buffer, 2.5 μl nucleotide mixture (DNTP), 16.5 μl deionized water, 0.5 μl Taq polymerase, 1 μl each of CampF primers (5'-AGAACACGCGGACCTATATA -3 ') and CampR (5'-CGATGCATCCAGGTAATGTAT-3') and component 6: DNA Ladder Mix molecular weight marker (Fermentas, Lithuania).

The use of the proposed group of inventions makes it possible to detect in TPF even traces of the presence of the main pathogenic representatives of the genus Campylobacter.

The invention is illustrated by the following figures: FIG. 1 shows an electrophoregram of the results of amplification of DNA isolated from a sample of patient M. with a temperature gradient from 53 ° C to 60 ° C, which shows that at a temperature of 60 ° C an amplification product corresponding to a size of 256 bp was formed; in fig. 2 - electrophoretogram of the results of amplification of the 16s RNA gene region of Campylobacter spp. 256 bp

The method is carried out as follows.

For PCR detection of Campylobacter spp. in tissue samples embedded in paraffin, the following stages are implemented: 1) preparation of sections of tissue samples embedded in paraffin, and dewaxing; 2) DNA extraction; 3) preparation of the reaction mixture; 4) amplification; 5) detection of results by horizontal electrophoresis in agarose gel.

1. From blocks of TFP, made from biopsies of the affected areas of the intestine, obtained during colonoscopy in patients with inflammatory bowel diseases, single-use scalpels prepare sections with a thickness of 1-2 mm (4-6 sections per 1 sample), place them in pre-labeled tubes type eppendorf, which are incubated in a portable incubator at 93 ° C until the paraffin is completely melted.

2. Without removing the tubes from the thermostat, quickly add 10 µl of component 1 from the kit to each sample with the analyzed sample, and suspend it on a vortex. Place the tubes in a portable thermostat (93 ° C) and, without removing, add 100 μl of component 2 from the kit to each. The tubes are then resuspended by vortexing and re-incubated in a portable incubator (93 ° C). Then each of the test tubes is placed in turn in a magnetic rack. Using a dispenser, the light phase (residues of paraffin and tissue detritus) is taken from the test tubes and discarded, without touching the sediment. Then, 200 μl of component 3 is added to each sample, mixed on a vortex for 10-15 seconds. Place the test tubes in a magnetic rack, then select and discard the transparent phase without touching the precipitate. Tubes with open lids are dried at 65 ° C for 5-10 minutes. Add 50 μl of component 4 to each tube, mix for 5-10 sec and place in a magnetic rack. The supernatant fluid containing the purified DNA is taken into clean tubes.

3. Preparation of the reaction mixture: take the required number of tubes for amplification of DNA of the test and control samples, mark. In each tube add 25 μl of component 5 from the kit, then - 3 μl of DNA sample obtained after extraction from the clinical and / or control sample; add 1 drop of mineral oil.

4. The prepared samples are placed in an MS-16 thermocycler "Tertsik" ("DNA-technology", Russia) or another thermal cycler. Amplification mode: denaturation (95 ° C, 1 min), then - 30 amplification cycles (DNA denaturation (95 ° C, 30 sec), annealing (60 ° C, 30 sec), elongation (72 ° C, 30 sec)) ...

5. The detection of the results of the reaction is carried out in horizontal electrophoresis in agarose gel. A 1.7% agarose gel is prepared in advance. The obtained amplifications in a volume of 10 μl are mixed with a leading dye, introduced into the wells of an agarose gel and connected to an electric current source. Electrophoresis is carried out in 110 V mode for 40 minutes. The result of the reaction is detected using a UV transilluminator and photographed. To control the size of the amplicons, a DNA Ladder Mix molecular weight marker (Fermentas, Lithuania) is used. After the end of electrophoresis, the gel is stained with ethidium bromide and photographed on a Gel Camera System (UVP, Inc. USA). The results are detected by comparing the size of the DNA sample under study with the DNA Ladder Mix molecular weight marker (Fermentas, Lithuania). If the size of the expected product is 256 bp, then these samples are recorded as containing DNA from Campylobacter spp. In contrast, in samples free of Campylobacter spp. after PCR, the amplification product is not detected.

The essence of the invention is illustrated by the following examples.

Example # 1.

Patient M., 54 years old. Diagnosis: nonspecific ulcerative colitis in the acute stage. Complaints about bloody stools, severe pain in the abdominal region, fever, fever. Test material: paraffin blocks of large intestine biopsies measuring 1.5 × 1.0 × 0.5 cm, No. 7.

From the TPF biopsy specimen of the large intestine measuring 1.5 × 1.0 × 0.5 cm, obtained from the affected areas of the intestine during colonoscopy, 5 sections with a thickness of 1–2 mm were prepared with disposable scalpels, placed in Eppendorf tubes, marked and incubated at 93 ° C until the paraffin is completely melted.

Without removing the tubes from the thermostat, 10 μL of component 1 from the kit was quickly added to each sample with the analyzed sample and suspended on a vortex. Continuing the incubation at 93 ° C, 100 μl of component 2 from the kit was added. Resuspended on a vortex and incubated at 93 ° C. The tube was placed in a rack with a magnet. Using a pipette, the light phase (residues of paraffin and tissue detritus) was taken from the tubes and discarded without touching the sediment from component 1. Then 200 μl of component 3 was added to each sample, resuspended on a vortex for 10-15 seconds. We placed the test tube in a rack with a magnet, after which the transparent phase was removed and discarded, without touching the sediment. The tube with the lid open was dried at 65 ° C for 10 min. 50 μl of component 4 was added to a test tube, mixed for 10 sec and placed in a magnetic rack. We took the supernatant containing the purified DNA into a clean test tube. Next, 25 μl of component 5 from the kit was introduced into the test tube, then 3 μl of DNA sample, and 1 drop of mineral oil was added.

The prepared samples were divided into 8 parts, each of which was amplified in an MS-16 "Tertsik" amplifier ("DNA-technology", Russia), changing the annealing temperature in a gradient from 53 ° C to 60 ° C to select the optimal one. Amplification mode: denaturation (95 ° C, 1 min), then - 30 amplification cycles (DNA denaturation (95 ° C, 30 sec), annealing (60 ° C, 30 sec), elongation (72 ° C, 30 sec).

The detection of the reaction results was carried out in horizontal electrophoresis in 1.7% agarose gel. The obtained amplicates in an amount of 10 μl are introduced into the wells of an agarose gel and connected to an electric current source. Electrophoresis is carried out in 110 V mode for 40 minutes. The result of the reaction is detected using a UV transilluminator and photographed. To control the quality of electrophoresis, a DNA Ladder Mix molecular weight marker (Fermentas, Lithuania) is used. After the end of electrophoresis, the gel is stained with ethidium bromide and photographed on a Gel Camera System (UVP, Inc. USA). The results are detected by comparing the size of the analyzed DNA sample with the DNA Ladder Mix molecular weight marker (Fermentas, Lithuania).

Conclusion. The optimum temperature for annealing to obtain the expected product was 256 bp. at 60 ° C (Fig. 1). The presence of pathogenic representatives of the genus Campylobacter in the examined tissue sample is determined. C. jejuni and / or C. coli and / or Cupsaliensis.

Example # 2.

Investigated in paraffin blocks biopsies of the affected areas of the colon obtained during colonoscopy of 9 patients with a diagnosis of ulcerative colitis (samples 1-9) and 8 patients with a clinical diagnosis of Crohn's disease (samples 10-17).

From them, single-use scalpels were used to prepare sections with a thickness of 1-2 mm (4-6 sections per sample) and placed them in Eppendorf tubes, marked and incubated at 93 ° C until the paraffin was completely melted.

Without removing the tubes from the thermostat, 10 μL of component 1 from the kit was quickly added to each sample with the analyzed sample and suspended on a vortex. We placed the tubes in a portable thermostat (93 ° C) and, without removing, added 100 μl of component 2 from the kit to each. The tubes were then resuspended on a vortex and re-incubated in a portable incubator (93 ° C). Next, each of the test tubes was placed in turn in a rack with a magnet. Using a pipette, the light phase (residual paraffin and tissue detritus) was taken from the test tubes and discarded, without touching the sediment. Then, 200 μl of component 3 was added to each sample, mixed on a vortex mixer for 10-15 seconds. We placed the test tubes in a rack with a magnet, after which the transparent phase was taken and discarded, without touching the sediment. The tubes with the lid open were dried at 65 ° С for 5-10 min. 50 μl of component 4 was added to each tube, mixed for 5-10 sec and placed in a magnetic rack. We took the supernatant containing the purified DNA into clean tubes.

Preparation of the reaction mixture: the required number of tubes for amplification of the DNA of the studied samples were taken, marked. Firstly, 25 μl of component 5 from the kit was introduced into each tube, then 3 μl of DNA of the test samples obtained after extraction from paraffin-embedded biopsies; added 1 drop of mineral oil.

The prepared samples were placed in an MS-16 "Tertsik" thermocycler ("DNA-technology", Russia) or another thermal cycler. Amplification mode: denaturation (95 ° C, 1 min), then - 30 amplification cycles (DNA denaturation (95 ° C, 30 sec), annealing (60 ° C, 30 sec), elongation (72 ° C, 30 sec).

The detection of the reaction results was carried out in horizontal electrophoresis in 1.7% agarose gel. The obtained amplicates in an amount of 10 μl were introduced into the wells of an agarose gel and connected to an electric current source. Electrophoresis was performed at 110 V for 40 min. To control the size of the amplicons, a DNA Ladder Mix molecular weight marker (Fermentas, Lithuania) was used. The result of the reaction was taken into account using a UV transilluminator and photographed. The results were detected by comparing the size of the studied DNA sample with the DNA Ladder Mix molecular weight marker (Fermentas, Lithuania). If the size of the expected product is 256 bp, then these samples were recorded as containing DNA from Campylobacter spp. In contrast, in samples free of Campylobacter spp. after PCR, the amplification product was not detected.

As a result of the studies carried out in biopsies obtained by colonoscopy and embedded in paraffin from 9 patients with a diagnosis of ulcerative colitis (samples 1-9), Campylobacter spp. DNA was found in samples 1, 3-9. Sample # 2 is negative. In paraffin-embedded biopsies of 8 patients with a clinical diagnosis of Crohn's disease (samples 10-17), Campylobacter spp. DNA was found in samples 11-17. Sample # 10 is negative (Fig. 2).

Claims (2)

1. A method for detecting bacteria of the genus Campylobacter in patients with inflammatory bowel diseases, including the isolation of DNA from a biological sample, presumably containing a pathogen, PCR in the presence of specific primers, amplification, characterized in that paraffin-embedded samples of intestinal tissue of patients are used as a biological sample with inflammatory bowel diseases, of which 4-6 sections with a thickness of 1-2 are prepared, the sections are dewaxed, then lysis, for which magnetic particles and a binding buffer for lysis are added, containing 4M guanidine thiocyanate, 100 mM Tris-HCl buffer, 2 mM ethylenediaminetetraacetate ( EDTA), 2% Triton X-100, after which alcohol precipitation and elution of the isolated DNA are carried out; for this, a wash solution containing 70% ethanol, 20 mM NaCl, 10 mM Tris-HCl, 2 mM EDTA is added, after drying, 50 μl elution buffer containing 10 mM Tris-HCl, 1 mM EDTA, PCR is performed using CampF primers

and CampR

to detect the amplification product, electrophoresis in agarose gel is carried out, and in the presence of an amplification product of 256 nucleotide pairs, the presence of pathogenic representatives of the Campylobacter genus C. jejuni and / or C. coli and / or C. upsaliensis is determined in the studied tissue sample. 2. A kit for the detection of bacteria of the genus Campylobacter in patients with inflammatory bowel diseases, including a binding buffer for lysis containing guanidine thiocyanate, Tris-HCl buffer and EDTA, an elution buffer containing Tris-HCl, and a reaction mixture containing Taq polymerase, water, a mixture of nucleotides and primers, characterized in that it additionally contains 10 μl of a suspension of magnetic particles, a wash solution containing 70% ethanol, 20 mM NaCl, 10 mM Tris-HCl (pH 7.4), 2 mM EDTA (pH 8.0), a marker molecular weights of DNA Ladder Mix, while the binding buffer for lysis contains 4M guanidine thiocyanate, 100 mM Tris-HCl buffer (pH 7.4), 2 mM EDTA (pH 8.0), 2% Triton X-100, elution buffer contains 10 mM Tris- HCl (pH 7.4), 1 mM EDTA (pH 8.0), and the reaction mixture contains 2.5 μL Taq buffer, 2.5 μL nucleotide mixture, 16.5 μL deionized water, 0.5 μL Taq polymerase, 1 each μl of CampF primers

and CampR

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