RU2483114C1 - METHOD TO DETECT GENES THAT DETERMINE ADHESINS, HEMOLYSINS, MANNOSE-RESISTANT HAEMAGGLUTININS IN GENITAL STRAINS OF Escherichia coli - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: method is proposed, including extraction of a biomaterial, extraction of a bacterial DNA, amplification of DNA fragments in the examined strain by PCR method with application of a unique set of oligonucleotide primers, where products of amplification are separated by means of electrophoresis in agarose gel. The PCR results determine simultaneously all three genes, which are taken into account by visualisation through dyeing with etidium bromide. Availability of pathogenicity genes in the examined biomaterial in strains Escherichia coli is determined by dimensions of produced DNA fragments: fim A - 248 base pairs, hly A,B,C - 233 base pairs, pap C - 251 base pairs.

EFFECT: invention may be used in medicine to detect genital coliform bacilli with pathogenicity factors by method of molecular hybridisation.

2 tbl, 3 dwg

Description

The invention relates to microbiology, genetic engineering, biotechnology and can be used to detect, by molecular hybridization, genital Escherichia coli with pathogenicity factors — adhesins, hemolysins, mannose-resistant hemagglutinins.

In recent decades, Escherichia coli has become a common nosocomial pathogen. Escherichia coli, as a representative of opportunistic microflora, is the cause of the development of inflammatory diseases of the urogenital tract.

The increase in virulence among heterogeneous populations of potentially pathogenic enterobacteria is currently an important object of research in various branches of biological and medical sciences. Due to the “spasmodic” nature and cardinality of the occurring transformations of the microorganism, the most significant changes in the pathogenicity of conditionally pathogenic enterobacteria are genetic recombinations that determine the genomic plasticity of microbes through several specific mechanisms related, in particular, to “islands” and “islands” pathogenicity associated with DNA fragments including discrete virulence genes [1]; [2].

In particular, among pathogens serovars Escherichia coli pathogenic for humans, “pathogenicity islands” were found that determine the key stages of the interaction of any pathogen with a macroorganism, including adhesion, production of toxins, and ability to withstand non-specific resistance factors [3].

Uropathogenic Escherichia coli, UPEC-uropathogenic E. coli, may include genes that control the synthesis of fimbrial adhesins (P and S), α-hemolysin (hly) and cytotoxic necrotizing factor-1 (cnf-1), the integration sites of which are the loci of the selenocysteine gene (sel), phenylalanine of a (phe) -specific tRNA [3]. There are known “pathogenicity islands” that control the synthesis of proteins associated with the absorption of iron ions necessary for the propagation of the pathogen in the tissue, as well as the genes of the type III secretion system, which is responsible for the one-stage transport of effector molecules from a bacterial cell to the cytoplasm of a eukaryotic cell with subsequent modification of the cytoplasmic proteins of affected targets [4].

In this regard, in some cases, the detection of genetic determinants, supplemented by the phenotypic characteristic of the expressed pathogenicity factor, makes it possible to objectively judge the etiological agent of the disease, which is important in the laboratory diagnosis of pathologies of the urogenital tract.

Currently, the most promising are molecular methods based on the detection of DNA of pathogens in the studied material, in particular, methods based on PCR technology. These methods have high sensitivity and specificity and make it possible to detect the presence of a very small number of copies of the genetic sequences of microorganisms in the micro quantities of the test material in a short time. In addition, modern modifications of the polymerase chain reaction, carried out in real time, also allow you to determine the quantitative values of microorganisms.

A known method for determining the frequency of occurrence of genetic determinants of virulence among E. coli strains isolated in children with an infection of the urinary system [2]. This method made it possible to establish the fact that the clinical form of urinary tract infection directly depends on the frequency of occurrence of uropathogenicity markers hly A, cnf-1, pap C, sfa G.

However, the described method is recommended by the authors only to identify the pathogen in the urine and is not intended to detect E. coli in other types of biological material (vaginal discharge, cervical canal).

There is a method for identifying the genetic determinants of the Escherichia pathogenicity factors that provide adhesion and toxin formation under conditions of associative symbiotic interaction of E. coli with B. hominis in vitro [5]. As a result of this study, a high frequency of occurrence of combinations of the pap C, pap H, sfa A, hly B and pap C, fim A, ehx genes was noted, which indicates their significance in the processes of adaptation to changing conditions. The described method is recommended by the authors to identify the causative agent in feces in case of intestinal infections against the background of blastocystic invasion.

The disadvantage of this method is the determination of DNA regions responsible for virulence factors, not in the "multiplex" system, i.e. not at the same time.

A known method for the determination of mutant thermolabile enterotoxin E. coli [6]. This method discloses an isolated polypeptide comprising the mutant A subunit of Escherichia coli thermolabile enterotoxin (LT), which has only one mutated residue, which is K, R, H or Y, compared to a wild-type thermolabile enterotoxin containing a specific amino acid sequence. The polypeptide of the method can be used as an adjuvant.

This method describes the isolation of only one pathogenicity factor - thermolabile enterotoxin.

The objective of the invention is to provide a method for identifying genes that determine adhesins, hemolysins, mannose-resistant hemagglutinins in genital strains of Escherichia coli, providing a technical result consisting in increasing the detection efficiency of the mentioned pathogenicity genes, the possibility of simultaneously identifying all three pathogenicity genes by unifying the amplification program by selection of primers with the same annealing temperature.

The technical result is achieved by the selection of biomaterial, the isolation of bacterial DNA, the amplification of DNA fragments of the studied strain by polymerase chain reaction using a unique set of oligonucleotide primers having nucleotide sequences:

5'-TGG-CTG-CCG-CAC-TAT-TCG-CC-3 ',

5'-TAT-TGG-GAC-CAC-GCG-TGS-CG-3 ',

5'-ACG-CCG-GCC-TTA-ACA-GTG-GC-3 ',

which have a high specificity of interaction with DNA fragments, the amplification products are separated by agarose gel electrophoresis, and the results of PCR reveal all three genes at the same time and take into account visualization by staining with ethidium bromide; in the presence of genes determining adhesins, hemolysins, mannose-resistant hemagglutinins in genital Escherichia coli strains in the studied biomaterial, DNA fragments fim A with a size of 248 bp, hly A, B, C - 233 bp, are formed, Rar C - 251 bp

The invention is illustrated by electrophoregrams of amplicons of the studied strain with molecular weight markers.

The results of the analysis are presented in figures 1-3.

Figure 1 shows the electrophoregram of amplification products obtained by PCR analysis of a DNA fragment of the Escherichia coli strain for the fim A gene, where 1-10 are Escherichia coli strains isolated from clinical material; 11 is a molecular weight marker.

Figure 2 shows the electrophoregram of amplification products obtained by PCR analysis of the Escherichia coli DNA fragment for the hly gene A, B, C, where 1-10 are Escherichia coli strains isolated from clinical material; 11 is a molecular weight marker.

Figure 3 shows the electrophoregram of the amplification products of the Escherichia coli DNA fragment for the rar C gene, where 1-13 are Escherichia coli strains isolated from clinical material; 14 - negative control; 15 is a molecular weight marker.

The method is as follows.

The selection of biomaterial (detachable vagina, cervical canal) is as follows. The vaginal discharge is taken with Volkman's sterile spoon from the posterolateral vaginal arches when viewed in the mirrors. Material from the cervical canal containing epithelial cells is obtained using a special urogenital probe with a brush after preliminary removal of visible mucus from the cervix with a sterile gauze swab. A culture study of the contents of the posterior and lateral arches of the vagina and cervical canal on enterobacteria is carried out. The studied biomaterial is seeded on a nutrient medium Endo or Levine. Crops are placed in a thermostat for 24-48 hours at a temperature of 37 ° C. On Endo medium, lactose-positive Escherichia coli grow in the form of flat-convex colonies of dark red color with a metallic sheen. On Levin's medium, Escherichia coli colonies are dark blue, with a metallic sheen, sometimes pinkish. Lactose-negative colonies are colorless. After incubation, identification is made on the basis of morphological, tinctorial (negative Gram stain) properties.

Next, bacterial DNA is isolated according to a standard method using a set of reagents for DNA extraction from a bioassay - “GS Test” (set No. 1).

Oligonucleotide primers used for PCR amplification of fragments of the fim A, hly A, B, C, rar C genes were selected on the basis of the nucleotide sequences of these genes in the Escherichia coli strain presented in the GenBank NCBI (CShA) database, EMBL (European Molecular biological library) and DDBJ (Japanese database of nucleotide sequences), as well as by sequencing a similar plot in strains isolated on the territory of the Russian Federation.

As a result of the alignment of the nucleotide sequences of the fim A, hly A, B, C, rar C genes of Escherichia coli (GenBank NCBI: CP001509.1, CP000819.1, CP001665.1, AM946981.1; M10133.1, CP000247.1, AM690760. 1; CP001509.1) and Escherichia coli strains isolated on the territory of the Russian Federation, their consensus sequence was selected and a unique set of oligonucleotide primers for the fim A, hly A, B, C, pap C genes with high specificity of interaction with DNA fragments was obtained :

The oligonucleotides of the primers and probe were synthesized by the amidophosphite method (Syntol CJSC, Moscow; passport No. 3873).

Characterization of oligonucleotide primers for the fim A, hly A, B, C, and Cp sites is presented in Table 1.

Table 1 Parameter Characteristic Gene fim A Direct primer (f) 5'-3 ' TGG-CTG-CCG-CAC-TAT-TCG-CC Reverse Primer (r) 5'-3 ' TGC-AAC-GGC-AGC-ATT-AGC-GG The estimated melting temperature of the forward primer + 60.0 ° C The estimated melting temperature of the reverse primer + 60.0 ° C Theoretical specificity All available to study the strain of E. coli The length of the amplified plot (bp) 248 Hly gene A, B, C Direct primer (f) 5'-3 ' TAT-TGG-GAC-CAC-GCG-TGS-CG Reverse Primer (r) 5'-3 ' CGC-CAT-CTC-CGC-CGT-TCA-GG The estimated melting temperature of the forward primer + 60.0 ° C The estimated melting temperature of the reverse primer + 60.0 ° C Theoretical specificity All E. coli strains available for study The length of the amplified plot (bp) 233 Gene pap C Direct primer (f) 5'-3 ' ACG-CCG-GCC-TTA-ACA-GTG-GC Reverse Primer (r) 5'-3 ' CA G-TCA-CCA-CGC-CCG-TTS-SS The estimated melting temperature of the forward primer + 60.0 ° C The estimated melting temperature of the reverse primer + 60.0 ° C Theoretical specificity All E. coli strains available for study The length of the amplified plot (bp) 251

The polymerase chain reaction is carried out according to standard technology.

The optimal parameters of the amplification reaction with oligonucleotide primers and probe were selected. In particular, the reaction mixture has the following composition: 0.5 mM Tris-Cl buffer, pH 8.6; 0.5 M KCl; 15 mM MgCl ₂ ; 2.5 mM deoxynucleotide triphosphates; 0.2 mM forward and reverse primers; Taq DNA polymerase - 50 μl.

PCR can be performed, for example, in a Tercik device (NPO DNA-Technology, Russia) using the program indicated in Table 2.

table 2 For amplifiers with active regulation (by solution in vitro): Tertsik (NPO DNA-Technology, Russia) Cycle number Step Temperature Duration Number of repetitions one one 95 ° C 5 minutes one 2 one 95 ° C 10 sec 2 60 ° C 20 sec 35 3 72 ° C 20 sec 3 one 72 ° C 2 minutes one

The threshold cycle values are determined automatically by the device.

PCR results are recorded by electrophoresis of amplification products on an agarose gel stained with ethidium bromide, for which ready-made agarose gels containing 2-3% agarose and ethidium bromide are used. Electrophoregram analysis is performed using the Gel Imager video system and Gel Analyzer software.

PCR results simultaneously determine all three pathogenicity genes and take into account by visualizing PCR staining with ethidium bromide in ultraviolet light.

In the case of a positive reaction to Escherichia coli, a 248 bp DNA fragment fim A appears in electrophoresis, 233 bp hly A, B, C, and 251 bp rar C. When introducing into the reaction of DNA of other types of microorganisms specific bands at the level where the marker strip is located with the corresponding length of the amplified region, during PCR was not observed.

The invention is illustrated by the following example.

To test the proposed method used preparations of total DNA isolated from clinical samples of biomaterial (vaginal discharge, cervical canal) taken from women of reproductive age with various inflammatory diseases. A total of 13 samples were prepared.

Isolation of DNA from 100.0 μl of the prepared material was performed using kit No. 1. The uniformity and transparency of all solutions were preliminarily checked. In the lysing and washing solution No. 1, precipitation of crystals is allowed, since the entire set of reagents is stored at 2-8 ° C. Before starting work, the crystals must be dissolved by heating the vials for 12-20 minutes at 50 ° C.

Tubes with samples containing the analyzed material in 0.5 ml of sterile physiological saline were centrifuged for 10 minutes at 13,000 rpm to obtain a precipitate. The supernatant was removed using a trap flask, leaving about 100 μl above the pellet.

Simultaneously with the isolation of DNA from biological material, a negative control sample was prepared. For this, 100 μl of sterile physiological saline was added to a separate plastic tube with a volume of 1.5 ml.

To process several samples, taking into account the negative control, 150 μl were mixed in a separate tube per sample of the lysis solution and 20 μl per sample of the previously suspended sorbent. Then, 170 μl of the resulting mixture was added to each tube with the sample and to the tube with a negative control, and all tubes were vortexed for 3-5 seconds to obtain a uniform suspension of the pellet with the lysing mixture. After this, the tubes were placed in a solid state thermostat for 20 minutes at 50 ° C.

After incubation, the tubes were centrifuged for 1 minute at 13,000 rpm. Using a flask trap, the supernatant was completely removed, being careful not to touch the sediment. Then, 200 μl of Wash Solution No. 1 was added to the pellet, the tubes were vortexed for 3-5 seconds and centrifuged for 13 minutes at 13,000 rpm. After this, the supernatant was removed from each tube, 200 μl of Wash Solution No. 2 was added, the tubes were vortexed vigorously for 3-5 seconds and centrifuged for 13 minutes at 13,000 rpm.

After centrifugation using a trap flask, the supernatant was removed and 200 μl of Wash Solution No. 3 was added to each tube. Vortex tubes were shaken for 3-5 seconds and centrifuged for 1 minute at 13,000 rpm. The supernatant was removed and all tubes with open caps were thermostated for 5 minutes at 50 ° C to dry the pellet.

After the pellet became dry under visual control, 100 μl of elution solution was added and the tubes were vortexed for 5-10 seconds.

After obtaining a uniform suspension, the tubes were thermostatically controlled for 5 minutes at 50 ° C.

At the end of the incubation, the tubes were centrifuged for 1 minute at 13,000 rpm. If the sample needed to be stored for a long time, then part of the supernatant containing DNA was taken into a new tube with the appropriate labeling. The rest was ready to be added to the reaction mixture for amplification.

DNA amplification reaction

The reagent kit for the amplification reaction was previously thawed at room temperature.

In a tripod "workstation" was placed a microtube with a volume of 0.5 ml and labeled them in accordance with the research protocol. Then, in a separate tube per 1 sample, the reaction mixture was prepared.

In particular, the reaction mixture has the following composition: 0.5 mM Tris-Cl buffer, pH 8.6; 0.5 M KCl; 15 mM MgCl ₂ ; 2.5 mM deoxynucleotide triphosphates; 0.2 mM forward and reverse primers; Taq DNA polymerase - 50 μl.

On top of the reaction mixture was applied 1 drop of mineral oil to prevent drying. Then, 5 μl of a DNA solution of samples prepared at the stage of sample preparation was added under a layer of oil. First, a negative control sample was added, and then samples in accordance with the protocol. The optimal parameters of the amplification reaction with oligonucleotide primers and probe were selected (Table 2).

After the end of the reaction (on the day of the study or after storage in the morning of the next day), the tubes were rearranged in a support for detection tubes and delivered to the PCR product analysis room, which was carried out by the method of horizontal electrophoretic separation of DNA fragments in an agarose gel.

Electrophoretic analysis of PCR products

The contents of the Tris-borate electrophoresis buffer package (kit No. 3) were placed in a 1000 ml measuring cylinder, distilled water in the amount of 700 ml was added and thoroughly mixed until completely dissolved. Then the total volume of the buffer was adjusted to 1000 ml. The finished buffer was stored in a container with a tight-fitting lid at room temperature and was used for 1 week.

After preparing the buffer, the electrophoresis chamber was prepared for work: it was filled with buffer and a plate with agarose gel was placed between the compartments so that the buffer layer covered the plate by 3-5 mm. Then, 7.0 μl of the amplification product from each tube was carefully added to the corresponding wells of the gel. A molecular weight marker was added to a separate well to determine the size of the amplification. Then, a cover was mounted on the camera, the electrodes were connected, and a direct current source was turned on. Electrophoresis was carried out in the direction from the cathode (-) to the anode (+) for 15 minutes at an electric field strength of 10–20 V / cm (with a chamber width of 10 cm, the voltage was 200 V).

After electrophoresis, the direct current source was turned off, the lid was removed from the chamber, the plate with agarose gel was carefully removed and placed on a glass of the transilluminator.

A protective screen was installed, the transilluminator was turned on, and the results obtained were analyzed. The amplification product was visible in ultraviolet light (wavelength 230-260 nm) in the form of a luminous strip of red-orange color.

For documentation, the gel was recorded using a video system connected to a personal computer equipped with the corresponding Gel Explorer program. The results were taken into account according to the calculated lengths of the amplification products of the genes fim A, hly A, B, C, and C. Using the Gel Analizer program, a computer analysis of the obtained electrophoregrams was carried out with the aim of more accurate estimation of amplicon sizes taking into account calibration by molecular weight marker. All genes that determine the pathogenicity factors, fim A, hly A, B, C, rar C are determined simultaneously.

PCR results are recorded by electrophoretic separation of amplification products on an agarose gel stained with ethidium bromide.

The sizes of the generated gene fragments are determined using the primer systems fim A, hly A, B, C, and C. They are 248 bp, 233 bp, 251 bp, which corresponds to the size of the gene fragments in genital strains Escherichia coli. Samples containing a specific band at the level where the marker strip is located with the corresponding length of the amplified section are considered positive.

Thus, the claimed method allows you to quickly, efficiently and reliably identify genes that determine adhesins, hemolysins, mannose-resistant hemagglutinins of genital strains of Escherichia coli using primer systems fim A, hly A, B, C, rar C in samples of biological material.

In addition, the determination of these gene regions proceeds in the “multiplex” system, i.e. at the same time, which is achieved by selecting primers with the same annealing temperature of 60 ° C and allows to unify the amplification program.

LITERATURE

1. Johson J.R. Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise // J. Infect. Dis. - 2000. - Vol. 181. - P.261-272.

2. Bondarenko B.M., Haliullina SV., Fialkina S.V. et al. Markers of uropathogenicity of E. coli in children with an infection of the genitourinary system // Children's infections. - Number 3. - 2004. - P.21-23.

3. Mavzyutov A.R., Fialkina S.V., Bondarenko V.M. “Islands” of pathogenicity of conditionally pathogenic enterobacteria // ZhMEI. - No. 6. - 2002. - P.5-9.

4. Bondarenko V.M. "Islands" of pathogenicity of bacteria // Zh. microbiol. - No. 4. - 2001. - P.67-74.

5. Krasnoperova Yu.Yu. Characterization of changes in the pathogenic potential of symbiont microorganisms in protozoal-bacterial associations: Monograph. - Moscow: Flint. - 2010.

6. RF patent No. 2441879, publ. 02/10/2012.

Claims (1)

A method for identifying genes determining adhesins, hemolysins, mannose-resistant hemagglutinins in genital strains of Escherichia coli, including selection of biomaterial, isolation of bacterial DNA, amplification of DNA fragments of the studied strain by PCR using a unique set of oligonucleotide primers having nucleotide sequences:
5'-TGG-CTG-CCG-CAC-TAT-TCG-CC-3 ',
5'-TAT-TGG-GAC-CAC-GCG-TGS-CG-3 ',
5'-ACG-CCG-GCC-TTA-ACA-GTG-GC-3 ',
which have a high specificity of interaction with DNA fragments, and the amplification products are separated by agarose gel electrophoresis, and the results of PCR are determined simultaneously by all three genes and which are taken into account by visualizing staining with ethidium bromide; in the presence of genes that determine adhesins, hemolysins, mannose-resistant hemagglutinins in the studied biomaterial in the studied biomaterial of Escherichia coli strains, fim A DNA fragments with a size of 248 bp, hly A, B, C - 233 bp are formed, Rar C - 251 bp

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