RU2443772C1 - Set of francisella tularensis bacteria strains for production of reference dna preparation set, set of dna preparations for genetically diagnostic research - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: invention represents reference strains Francisella tularensis of tularensis subtype, including two genetic groups AI and AII, of holartica subtype, including biovars japonica, erythromycin-sensitive and erythromycin-resistant, Ery^s and Ery^R, and of mediasiatica subtype. Strains are deposited in the State Collection of Pathogenic Microbes "Microbe". All strains are natural, apart from the strain of holartica subtype, biovar japonica, which is extracted from a human being. Strains are produced by selection of clones that stably preserve phenotypic properties and containing genes, nucleotide sequence of which is specified for the type and subtype. The set of reference DNA preparations is created on the basis of these strains and may be used for genetic and immunological research.

EFFECT: using the invention will make it possible to increase efficiency of diagnostic research, to standardise and to increase quality of diagnostic preparations at production stages.

4 cl, 4 dwg, 1 tbl, 10 ex

Description

The invention relates to medical microbiology and can be used in the diagnosis of the causative agent of especially dangerous infections, namely the determination of the type and subspecies of the causative agent of tularemia, as well as in the construction of genetic diagnostic preparations.

It is known that the causative agent of tularemia belongs to a subclass of protobacteria, the family Francisellaceae, the genus Francisella and is represented by species Francisella tularensis, Francisella novicida, Francisella philomiragia. Three subspecies are distinguished within the Francisella tularensis species: tularensis, holarctica, mediasiatica (1). The pathogenicity of tularemia microbe depends on its subspecies. The greatest virulence for humans is the subspecies tularensis (type A). The subspecies holarctica (type B) and mediasiatica are less virulent for humans. Differences in pathogenicity are one of the signs of differentiation of subtypes of tularemia microbe. The identification of the causative agent of tularemia in biological material and samples from environmental objects is carried out according to MU 3.1.2007-05 (2).

In a mass study, serological, immunological methods are used, for example, the reaction of neutralizing antibodies (RNA) with tularemia antigenic erythrocyte diagnosticum, enzyme-linked immunosorbent assay (ELISA), the sensitivity of which is 10 ⁴ -10 ⁵ MK in 1 ml or 5 ng / ml antigen. The duration of the study is up to 4-5 hours. The immunofluorescence method (RIF) with the same sensitivity allows the pathogen to be detected within 1 h. However, it is impossible to determine the pathogen subspecies using these methods. Bacteriological methods are not used to study material from environmental objects, since crops are contaminated with extraneous microflora. The most effective way to detect tularemia microbe is a biological sample. White mice infected with material containing the causative agent of tularemia die on 3-4, 7-9 days, depending on the virulence of the microbe. At the current level of diagnostic research, PCR analysis (polymerase chain reaction) is used to indicate and accelerate diagnostics, which, in terms of sensitivity and speed of obtaining the result, exceeds all the methods described above. However, identification of the causative agent of tularemia taking into account subspecies differentiation is not carried out, due to the lack of diagnostic drugs. Nevertheless, the wide movement from various countries of near and far abroad of cargo, transport, people with hand luggage engaged in commercial activities, may be the source of the emergence of the causative agent of tularemia of any subspecies in the territory of the Russian Federation. In this case, timely and accurate diagnosis of the causative agent of tularemia will contribute to the rapid resolution of epidemiological issues.

When studying the various properties of the tularemia pathogen, researchers use strains of the tularemia pathogen available for this region to compare.

The bacterial strain Francisella tularensis B399 A-CoLe Strr 2500 / K of the non-Arctic subspecies attenuated for the preparation of a live vaccine against tularemia infection is known (3). The strain was obtained by selection of streptomycin-resistant mutants of the tularemia microbe of strain B399 A-CoLe of non-Arctic subspecies.

It is known to use the collection of strains of Francisella tularensis for comparison when studying the biological properties of the population structure of the causative agent of tularemia (4, 5).

In the WHO documents, an attenuated strain of Francisella tularensis ATCC 6223 (B-38) is proposed as a control, which is recommended for comparison in determining the species of the pathogen, but it cannot be used as a control in determining all subspecies of tularemia (6).

To unify, standardize and ensure safety during molecular genetic studies (PCR analysis, biochips) in laboratories of various levels (territorial, regional, federal) it is advisable to use sterile DNA preparations for control, and not DNA obtained ex tempro from tularemia pathogen strains .

Control preparations for diagnostic kits are known for identifying pathogens of Chlamydia trachomatis, Ureaplasma urealyticum, Micoplasma hominis, and others in luminescent microscopy, produced by various companies, for example, the Galart research and production company, which is a prepared and fixed smear of the pathogen of infectious diseases. Control preparations are intended for internal control in laboratories performing diagnostic studies and comparative tests. The use of such control preparations eliminates the need to keep a collection of living pathogens of infectious diseases.

Control DNA preparations (molecular weight markers) for agarose gel electrophoresis are known, which are used for a rough estimate by comparing the intensity of fragments of the same size, and also as a standard for molecular weights (e.g., DNA of the Lambda phage) (7).

Closest to the solution are control samples of DNA and RNA manufactured by Vircell Microbiologists, which are lyophilized samples of genomic DNA and RNA of pathogens intended for use as an external positive control of qualitative and quantitative PCR (8).

According to the patent search and literature data, there is currently no information on the presence of control preparations of bacterial DNA of the causative agent of tularemia for molecular genetic studies.

The objective of the present invention is a set of reference strains of Francisella tularensis subspecies tularensis (type A), holarctica (type B) and mediasiatica stably preserving phenotypic properties containing genes whose nucleotide sequence is specific for the species, subspecies, biovar, and the creation of a set of control preparations based on them DNA for genetic diagnostic research.

The technical result obtained from the use of the invention is expressed in increasing the effectiveness of diagnostic studies by introducing control preparations that allow them to be used in laboratories of any level, including those that cannot store pathogens of pathogens of the I-II pathogenicity groups, to eliminate technical errors when performing genetic methods, as well as standardize and improve the quality of diagnostic products at the production stages.

To achieve the technical result, a set of reference strains of Francisella tularensis subspecies tularensis, including two genetic groups AI and AII, subspecies holarctica, including three biovars: Japonica, erythromycin-sensitive (Ery ^s ), erythromycin-resistant (Ery ^R ) and subspecies mediasiatica for genetic and , as well as a set of DNA preparations from them for genetic diagnostic studies.

The inventive strains were obtained by selection of clones stably preserving phenotypic properties and containing genes whose nucleotide sequence is specific for the species and subspecies.

Characterization of strains:

The strain Francisella tularensis of the subspecies mediasiatica (A-61 (117)) KM 4 is natural, isolated in Uzbekistan (Karakalpak Autonomous Soviet Socialist Republic) in 1960 from ixodid ticks taken from cows.

Cultural and morphological characters.

Cocci or coccobacilli are small, ranging in size from 0.4 to 0.7 microns. On FT agar, pH 7.2 after 24 hours, colorless or grayish, smooth medium-sized colonies with smooth edges are formed. In a liquid nutrient medium, growth on the surface of the medium.

Physiological and biochemical characteristics.

Aerobe. The optimum growth temperature is 37 ° C. The optimum pH is 7.0-7.4. Auxotrof, requires the presence in the environment of glucose, cystine (cysteine), vitamin B. It does not have penicillinase activity. It has citrulinureidase activity. Glycerin ferments to acid without gas, does not ferment D-maltose, D-glucose.

The strain Francisella tularensis subspecies mediasiatica agglutinates species diagnostic serum

The special properties of the strain.

The strain Francisella tularensis KM 4 of the mediasiatica subspecies contains species-specific ig IBC genes in the genome, pathogenicity islands FPI of the tularemia microbe, the RDI differentiation region is specific for the tularemia microbe of the Central Asian subspecies. The nucleotide sequence of the sdhA gene fragment of 521 bp of this strain is identical to that of strains of Francisella tularensis subspecies mediasiatica FSC 147, the full genome of which is presented in the NCBI Gen Bank database (CP.000915.1). It has nucleotides that are variable at the subspecies level: 80- "G", 96- "T", 365- "T" (nucleotide numbering within the fragment is 521 bp).

The strain Francisella tularensis subspecies holarctica biovar Ery ^s (21-L) KM 3-natural, isolated in Yakutia in 1976 from lemming.

Cultural and morphological characters.

Cocci or coccobacilli are small, ranging in size from 0.6 to 0.7 microns. On FT agar, pH 7.2 after 24 hours, colorless or grayish, smooth medium-sized colonies with smooth edges are formed. In a liquid nutrient medium, growth on the surface of the medium.

Physiological and biochemical characteristics.

Aerobe. The optimum growth temperature is 37 ° C. The optimum pH is 7.0-7.4. Auxotrof, requires the presence in the medium of glucose, cystine (cysteine), vitamin B. It has penicillinase activity, does not have citrulinureidase activity, does not ferment glycerin, ferments D-maltose, D-glucose.

The strain Francisella tularensis subspecies holarctica biovar Ery ^s agglutinates species diagnostic serum

The special properties of the strain.

The strain Francisella tularensis of the subspecies of the holarctica biovar Ery ^s KM 3 contains the species-specific ig IBC genes of the pathogenicity island FPI of the tularemia microbe, the RDI differentiation region specific for the tularemia microbe of the holarctic subspecies of the Ery ^s and Ery ^R biovars. The nucleotide sequence of the sdhA gene fragment of 521 bp of this strain is identical to that of the strains of Francisella tularensis holarctica biovars Ery ^s and Ery ^R LVS, OSU18, FTNF002-00, the full genome of which is presented in the NCBI Gen Bank database (AM 233362.1, CP.000437.1, CP.000803. 1). It has nucleotides that are variable at the subspecies level: 80- "G", 96- "C", 365- "T" (nucleotide numbering within the fragment is 521 bp).

The strain Francisella tularensis subspecies holarctica biovar Ery ^R (M-498) KM 8 natural, isolated in the Astrakhan region in 1989 from ordinary voles.

Cultural and morphological signs:

Cocci or coccobacilli are small, ranging in size from 0.6 to 0.7 microns. On FT agar, pH 7.2 after 24 hours, colorless or grayish, smooth medium-sized colonies with smooth edges are formed. In a liquid nutrient medium, growth on the surface of the medium.

Physiological and biochemical signs:

Aerobe. The optimum growth temperature is 37 ° C. The optimum pH is 7.0-7.4. Auxotrof, requires the presence in the medium of glucose, cystine (cysteine), vitamin B. It has penicillinase activity, does not have citrulinureidase activity, does not ferment glycerin, ferments D-maltose, D-glucose.

The strain Francisella tularensis subspecies holarctica biovar Egu ^R agglutinates species diagnostic serum

Special properties of the strain:

The strain Francisella tularensis of the subspecies of the holarctica biogar Egu ^R KM contains species-specific ig IBC genes in the genome of the pathogenicity island FPI of the tularemia microbe, the RDI differentiation region is specific for the tularemia microbe of the holarctic subspecies of the Ery ^s and Egu ^R biovars. The nucleotide sequence of the sdhA gene fragment of 521 bp of this strain is identical to that of the strains of Francisella tularensis holarctica biovars Ery ^s and Ery ^R LVS, OSU18, FTNF002-00, the full genome of which is presented in the NCBI Gen Bank database (AM 233362.1, CP 000437.1, CP 000803. 1). It has nucleotides that are variable at the subspecies level: 80- "G", 96- "C", 365- "T" (nucleotide numbering within the fragment is 521 bp).

The strain Francisella tularensis subspecies holarctica biovar japonica (Kosho) KM 5 natural, isolated in Japan in 1973 from humans.

Cultural and morphological signs:

Cocci or coccobacilli are small, ranging in size from 0.3 to 0.6 microns. On FT agar, pH 7.2 after 24 hours, colorless or grayish, smooth medium-sized colonies with smooth edges are formed. In a liquid nutrient medium, growth on the surface of the medium.

Physiological and biochemical signs:

Aerobe. The optimum growth temperature is 37 ° C. The optimum pH is 7.0-7.4. Auxotrof, requires the presence in the environment of glucose, cystine (cysteine), vitamin B. It has penicillinase activity, does not have citrulinureidase activity. Glycerin, D-maltose, D-glucose ferments to acid without gas.

The strain Francisella tularensis subspecies holarctica biovar japonica KM 5 agglutinates species diagnostic serum

Special properties of the strain:

The strain Francisella tularensis subspecies holarctica biovar japonica KM 5 contains in the genome species-specific ig IBC genes, pathogenicity islands FPI of the tularemia microbe, the RDI differentiation region specific for the tularemia microbe of the Holarctic subspecies of the Japanese biovar. The nucleotide sequence of the sdhA gene fragment of 521 bp of this strain is identical to that of strains of Francisella tularensis subspecies holarctica, LVS, OSU18, FTNF002-00, the full genome of which is presented in the NCBI Gen Bank database (AM 233362.1 CP.000803.1). It has nucleotides that are variable at the subspecies level: 80- "G", 96- "C", 365- "C" (nucleotide numbering within the fragment is 521 bp).

The strain Francisella tularensis of the subspecies tularensis (0-328) KM 7 (genetic group AI) is a natural strain isolated from ticks in Canada in 1935.

Cultural and morphological signs:

Cocci or coccobacilli are small, ranging in size from 0.3 to 0.7 microns. On FT agar, pH 7.2 after 24 hours, colorless or grayish, smooth medium-sized colonies with smooth edges are formed. In a liquid nutrient medium, growth on the surface of the medium.

Physiological and biochemical signs:

Aerobe. The optimum growth temperature is 37 ° C. The optimum pH is 7.0-7.4. Auxotrof, requires the presence in the medium of glucose, cystine (cysteine), vitamin B. It has penicillinase and citrulinureidase activity, glycerol, D-maltose, D-glucose ferments to acid without gas.

Agglutinated species diagnostic serum.

Special properties of the strain:

The strain Francisella tularensis subspecies tularensis KM 7 (genetic group AI) contains species-specific ig IBC genes, pathogenicity islands FPI of the tularemia microbe in the genome, the RDI differentiation region is specific for the tularemia microbe of the tularensis subspecies. The nucleotide sequence of the sdhA gene fragment of 521 bp this strain is identical to that of the strains of Francisella tularensis subsp.tularensis SCHU S4, FSC198, the full genome of which is presented in the NCBI Gen Bank database (AJ 749949.2.AM 286280.1). It has nucleotides that are variable at the subspecies level: 80- "A", 96- "C", 365- "T" (nucleotide numbering inside the fragment is 521 bp).

The strain Francisella tularensis of the subspecies tularensis (E-261 (B.399) KM 6 (genetic group AN) is a natural strain isolated in the United States in 1972 from a deer fly.

Cultural and morphological signs:

Cocci or coccobacilli are small, ranging in size from 0.3 to 0.7 microns. On FT agar, pH 7.2 after 24 hours, colorless or grayish, smooth medium-sized colonies with smooth edges are formed. In a liquid nutrient medium, growth on the surface of the medium.

Physiological and biochemical signs:

Aerobe. The optimum growth temperature is 37 ° C. The optimum pH is 7.0-7.4. Auxotrof, requires the presence in the medium of glucose, cystine (cysteine), vitamin B. It has penicillinase and citrulinureidase activity, glycerol, D-maltose, D-glucose ferments to acid without gas.

Agglutinated species diagnostic serum.

Special properties of the strain:

The strain Francisella tularensis of the subspecies tularensis KM 6 (genetic group AII) contains species-specific ig IBC genes, pathogenicity islands FPI of the tularemia microbe in the genome, the RDI differentiation region is specific for the tularemia microbe of the tularensis subspecies. The nucleotide sequence of the sdhA gene fragment of 521 bp this strain is identical to that of the strains of Francisella tularensis subsp.tularensis WY96-3418, the full genome of which is presented in the NCBI Gen Bank database (CP. 000608.1). It has nucleotides that are variable at the subspecies level: 80- "G", 96- "C", 365- "T" (nucleotide numbering within the fragment is 521 bp).

The inventive strains of Francisella tularensis are deposited in the "State collection of pathogenic bacteria" Microbe "(Federal State Research Institution RosNIPCHI" Microbe "), where they are assigned numbers:

Francisella tularensis subspecies mediasiatica - KM 4

Francisella tularensis subspecies holarctica biovar Ery ^s - KM 3

Francisella tularensis subspecies holarctica biovar Ery ^R - KM 8

Francisella tularensis subspecies holarctica biovar japonica - KM 5

Francisella tularensis subspecies tularensis (genetic group AI) - KM 7

Francisella tularensis subspecies tularensis (genetic group AII) - KM 6

The technical result is also achieved by creating a set of DNA preparations from bacteria strains of Francisella tularensis of various subspecies and biovars for molecular genetic studies.

The preparations are highly purified bacterial genomic DNA of each subspecies Francisella tularensis obtained by treatment with certain enzymes, followed by filtration purification. The resulting preparations are arranged in a kit consisting of test tubes and vials containing:

- a sufficient amount of purified genomic DNA of bacterial strains Francisella tularensis subspecies tularensis of the genetic groups AI and AII, taken in equal proportions;

- a sufficient amount of purified genomic DNA of bacterial strains Francisella tularensis subspecies holarctica biovar Ery ^s , biovar Ery ^R , biovar japonica, taken in equal proportions;

- a sufficient amount of purified genomic DNA of bacterial strains Francisella tularensis subspecies mediasiatica;

- a sufficient amount of purified genomic DNA of the bacterial strain Francisella tularensis subspecies holarctica biovar japonica;

- a sufficient amount of purified genomic DNA of the bacterial strain Francisella tularensis subspecies holarctica biovar Ery ^s ;

- a sufficient amount of purified genomic DNA of the bacterial strain Francisella tularensis subspecies holarctica biovar Ery ^R ;

- a sufficient amount of purified genomic DNA of the bacterial strain Francisella tularensis subspecies tularensis genetic group AI;

- a sufficient amount of purified genomic DNA of the bacterial strain Francisella tularensis subspecies tularensis genetic group AII;

- the distributing buffer.

The kit is designed to perform gene diagnostic tests in determining the subspecies Francisella tularensis, to control the specificity of the developed test systems, for scientific genetic research as a positive or negative control.

Using the kit greatly facilitates the implementation of molecular genetic methods, ensures their standardization, improves the quality of experimental and diagnostic studies.

The inventive collection of reference strains of Francisella tularensis of various subspecies and a set of preparations from them can be used:

1) as reference strains, as well as initial when creating a set of control DNA preparations from bacterial strains Francisella tularensis of various subspecies for genetic research;

2) in the production of PCR - test systems for identifying the causative agent of tularemia of each subspecies or set of primers for all of the above subspecies as a positive control;

3) in the production of PCR - test systems of closely related microorganisms to exclude cross-reactions as a control;

4) for scientific genetic studies of strains of the causative agent of tularemia during molecular typing using gene amplification, restriction and sequencing technologies as control preparations;

5) to assess specificity in the development of new diagnostic preparations based on oligonucleotide microarrays (DNA chips);

6) for internal quality control of diagnostic studies of licensed laboratories as control preparations;

7) as reference strains of bacteria Francisella tularensis of various subspecies to assess the specificity, sensitivity and activity of diagnostic tularemia sera and diagnostic immunoglobulin preparations of diagnostic tularemia in immunological reactions.

Example 1. Determination of affiliation of the studied cultures to the species Francisella tularensis.

In accordance with MU 3.1.2007-05 (2) and the WHO Guidelines (6), it was proposed to use PCR with primers based on the 16S rDNA gene (specific at the level of the genus Francisella) and tul4, fopA, ISFtu2, iglABCD genes to identify isolated tularemia microbe strains. (specific to the species Francisella tularensis).

For the study, a suspension of 2 test strains of tularemia microbe grown in solid nutrient medium was prepared in 2 ml of 0.9% sterile sodium chloride solution according to the industry standard turbidity sample of 10 units of GISK named after L.A. Tarasevich (OSO 42-28-59-85P), which corresponds to 5 × 10 ⁹ sq.m. Then, 10-fold dilutions were made in a 0.9% sterile solution of sodium chloride to a final concentration of 1 × 10 ⁵ m.k. / ml. The disinfection of microbial suspensions was carried out according to MU 1.3.2569-09 (9). To the prepared bacterial suspension with a volume of 4.5 ml was added 0.5 ml from a solution of sodium merthiolate 0.1% concentration (dilution 1: 1000) to a final concentration of 0.01% (dilution 1: 10000), warmed up at a temperature of 56 ° C within 30 minutes Then, 0.1 ml of bacterial suspension was taken into 1.5 ml microcentrifuge tubes, and 300 μl of a lysis solution containing 6 M guanidine thiocyanate was added. Heated at a temperature of 65 ° C for 15 minutes

DNA isolation was carried out using a DNA isolation kit - DNA Sorb B (ILS, Russia) or any other company. The work was carried out in accordance with the manufacturer's instructions.

PCR was carried out in accordance with WHO guidelines (2007) using primers based on 16S rDNA (F5-F11) (10) and using an experimental gene-amplification test system with primers based on iglBC genes. The genomic DNA of bacterial strains Francisella tularensis of the subspecies tularensis of the genetic groups AI and AII, taken in equal proportions, was used as a positive control.

Analysis was carried out using electrophoresis. The amplified product in a volume of 10 μl was added to the wells of a 2% gel. An agarose gel was prepared on 1 × TAE with ethidium bromide, the electrophoresis chamber was filled with a similar buffer. Electrophoresis was carried out in accordance with the instruction manual for the power source for 20-25 min, with a voltage gradient of 10 V / cm. The dye must go 2/3 of the length of the track. After the end of the process, the gel was washed in running water and placed on a UV transilluminator. Accounting and documentation of the results was carried out on a gel-documenting system GelDoc (BioRad). The stained DNA in the gel is viewed under ultraviolet radiation, for which a transilluminator with a wavelength in the range of 254-312 nm is used, fragments of which appear in the form of luminous pink-red stripes.

Interpretation of the results. The results of PCR analysis were evaluated by the presence or absence on the electrophoregram of a specific band of amplified DNA at a level corresponding to the band of positive control.

When conducting PCR with primers based on 16S rDNA on the electrophoregram (figure 1), the following fragments were recorded:

Sample 1. The studied strain No. 1 - fragment size 1104 bp

Sample 2. The studied strain No. 2 - a fragment of size 1104 bp

Sample 3. Positive control (genomic DNA of bacterial strains Francisella tularensis subspecies tularensis of genetic groups AI and AII) - a fragment of 1104 bp

Sample 4. Negative control - no fragment.

The results obtained indicate the belonging of the studied strains No. 1 and No. 2 to the genus Francisella.

When conducting PCR with primers based on iglBC genes on the electrophoregram (figure 1), the following fragments were recorded:

Sample 1. The studied strain No. 1 - a fragment of 268 bp

Sample 2. The studied strain No. 2 - a fragment of 268 bp

Sample 3. Positive control (genomic DNA of bacterial strains Francisella tularensis subspecies tularensis of genetic groups AI and AII) - a fragment of 268 bp

Sample 4. Negative control - no fragment.

The obtained results indicate the belonging of the studied strains No. 1 and No. 2 to the species Francisella tularensis.

Example 2. Determination of the subspecies of tularemia microbe by PCR.

In accordance with the WHO Guidelines (6), Ft-M19, ISFtu2, pdpD, and RD1 regions are used as target DNAs to determine subspecies of the tularemia microbe by PCR.

Sample preparation of bacterial suspensions of 2 test strains of tularemia microbe was carried out similarly as in example 1, bringing the concentration of microbial cells in 1 ml to 1 × 10 ⁷ . Sample disinfection and DNA isolation were carried out similarly as in example 1.

To identify the RD1 region, oligucleotide primers proposed by Broekhuijsen et al. [eleven]:

RD1-F 5 '- TTTATATAGGTAAATGTTTTACCTGTACCA-3'

RD1-R5 '- GCCGAGTTTGATGCTGAAAA-3'.

For PCR, 7 microtubes with a volume of 0.6 ml were prepared. In a separate 0.6 ml microtube, the general reaction mixture was prepared on the basis that 1 sample required: 10 × B - 2.5 μl, 50 mM MgCl ₂ solution - 1.5 μl, 25 mM dNTP - 0, 2 μl, primers RD1-F and RD1-R at a concentration of 100 pmol / μl - 0.15 μl each, Taq DNA polymerase enzyme with an activity of 5 units / μl - 0.4 μl, water free of nucleases (by filtration ) - 5.6 μl, final volume - 15 μl. The reaction mixture was thoroughly mixed in a microcentrifuge shaker and transferred to 15 μl in prepared tubes. 20-25 μl of mineral paraffin oil was layered on top. After that, 10 μl of the DNA of the studied cultures of F. tularensis were introduced into the first two tubes with separate tips with filters. Then, 10 μl of nuclease-free water, a negative control, was added to the last tube. After that, 10 μl of DNA preparations of tularemia microbe of various subspecies was added to tubes 3–6 — positive control. As a positive control, the genomic DNA of bacteria strains Francisella tularensis subspecies tularensis of the genetic groups AI and II, taken in equal proportions (3rd tube), the genomic DNA of a strain of the bacteria Francisella tularensis subspecies mediasiatica (4th tube), the genomic DNA of a strain of the bacteria Francisella tularensis subspecies holarctica biovar japonica (5th tube), genomic DNA of bacterial strains Francisella tularensis subspecies holarctica biovars Ery ^s and Ery ^R taken in equal proportions (6th tube). The tubes were placed in an amplifier and the reaction was carried out according to the following program: 95 ° C for 10 min; 30 cycles from 94 ° C - 30 s, 60 ° C - 1 min, 72 ° C - 1 min; 72 ° C - 3 min.

Analysis was carried out using electrophoresis. The amplified product in a volume of 10 μl was mixed with 2 μl of the leading dye (for example, 6 × Orange DNA Loading Dye, Fermentas, Cat No. R0631) and introduced into the wells of 3% NuSieve 3: 1 agarose gel. An agarose gel was prepared on 1 × TAE with ethidium bromide, the electrophoresis chamber was filled with a similar buffer. Electrophoresis was carried out in accordance with the instruction manual for the power source for 2.5-3 hours, with a voltage gradient of 5 V / cm. After the end of the process, the gel was washed in running water and placed on a UV transilluminator. Accounting and documentation of the results was carried out on a gel-documenting system GelDoc (BioRad). The stained DNA in the gel is viewed under ultraviolet radiation, for which a transilluminator with a wavelength in the range of 254-312 nm is used, fragments of which appear in the form of luminous pink-red stripes.

Interpretation of the results. The results of PCR analysis were evaluated by the presence or absence of a specific band of amplified DNA on the electrophoregram at the level of the corresponding bands of positive controls.

When conducting PCR with primers RD1-F and RD1-R, flanking RD1 is the region of tularemia microbe, the following fragments were recorded (figure 2):

Sample 1. The investigated strain No. 1 - fragment size 924 bp

Sample 2. The studied strain No. 2 - fragment size 1522 bp

Sample 3. Positive control - genomic DNA of bacterial strains Francisella tularensis subspecies tularensis of genetic groups AI and AII - fragment size 1522 bp

Sample 4. Positive control - genomic DNA of a strain of the bacterium Francisella tularensis subspecies mediasiatica - fragment size of 1453 bp

Sample 5. Positive control - genomic DNA of a strain of the bacterium Francisella tularensis subspecies holarctica biovar japonica - fragment size 1136 bp

Sample 6. Positive control - genomic DNA of strains of bacteria Francisella tularensis subspecies holarctica biovars Ery ^s and Ery ^R , taken in equal proportion, fragment 924 bp in size.

Sample 7. Negative control - no fragments.

The results indicate that the studied strain No. 1 belongs to Francisella tularensis subspecies holarctica of the biovars Ery ^s and Ery ^R , strain No. 2 belongs to Francisella tularensis subspecies tularensis.

Example 3. The use of reference strains of tularemia microbe of various subspecies to assess the specificity of the experimental test system for detecting Y.pestis DNA by PCR with hybridization-fluorescence real-time results.

For the study, we used an experimental test system for detecting Y. pestis DNA by PCR with hybridization-fluorescence real-time results taking into account primers based on a species-specific fragment of the plague pathogen chromosome. To assess the sensitivity and specificity of the test system, Y. pestis EV bacterial suspensions with a concentration of 1 × 10 ⁴ -1 × 10 ² m.k. / ml and F.tularensis subspecies tularensis of genetic group AI with a concentration of 1 × 10 ⁷ m.k. were used. ./ml. Sample preparation, disinfection and DNA isolation were carried out in the same way as in example 1. PCR staging and recording of results was carried out in accordance with the instructions for the drug.

The presence of specific fluorescence was noted only in the study of samples containing plague microbe DNA through the JOE channel (figure 4). When examining a sample containing the DNA of F.tularensis subspecies tularensis genetic group AI, a negative result was obtained. The presented data indicate the specificity of the developed experimental test system for detecting Y. pestis DNA by PCR with hybridization-fluorescence taking into account the results in real time.

Example 4. Determining the belonging of the causative agent of tularemia to a subspecies, biovar and genetic group by sequencing a fragment of the sdhA gene.

The preparation of the bacterial suspension, disinfection of the material and DNA isolation was carried out as described in example 1. For the study, we used a bacterial suspension of strains of tularemia microbe with a concentration of 1 × 10 ⁷ MK./ ml For analysis, primary PCR was performed with primers flanking 521 bp. a fragment of the sdhA gene. Sequencing of the amplicons obtained in PCR was performed on an AB1 PRISM 3100 Genetic Analiser automatic sequencer or any other company. Sample preparation for sequencing was carried out in accordance with the manufacturer's recommendations. As reference samples, reference strains of Francisella tularensis subspecies tularensis of the genetic groups AI and AII, the holarctica subspecies of the biovars Ery ^S and Ery ^R and japonica and the mediasiatica subspecies or genomic DNA preparations were used as reference samples for comparing nucleotide sequences. The results were evaluated using genetic programs (e.g., Mega 4.1) by aligning the nucleotide sequences and comparing the nucleotide composition of the studied strains and reference strains of tularemia microbe of different subspecies, biovars and genetic groups. The studied cultures of F.tularensis belong to a subspecies of tularensis of the genetic groups AI and AII at 100% homology of the studied fragment of the sdhA gene with that of reference strains of F.tularensis of the subspecies of tularensis of the genetic groups AI and AII. The studied cultures of F.tularensis belong to the subspecies of mediasiatica at 100% homology of the studied fragment of the sdhA gene with that of the reference strain F.tularensis of the subspecies of mediasiatica. The studied cultures of F.tularensis belong to the holarctica subspecies of the biovars Ery ^s and Ery ^R at 100% homology of the studied fragment of the sdhA gene with that of reference strains of F.tularensis subspecies holarctica to the biovars Ery ^s and Ery ^R. The studied cultures of F.tularensis belong to the subspecies of holarctica biovar japonica at 100% homology of the studied fragment of the sdhA gene with that of the reference strain F.tularensis subspecies holarctica of the biovar japonica.

Example 5. The assessment of the specificity of diagnostic test systems based on oligonucleotide microarrays (DNA chips).

Multiparameter analysis on biochips, using reference strains of F.tularensis or a set of genomic DNA preparations from them, includes the following steps: preparing a sample for analysis, performing asymmetric multiplex PCR (amPCR), hybridization on a biological microchip, and taking into account the results of the analysis.

The preparation of strains (analyzed and reference) is carried out in accordance with MU 1.3.2569-09 (9).

DNA isolation for subsequent PCR analysis is carried out using a DNA isolation kit in accordance with the manufacturer's instructions.

Amplification of the isolated DNA is carried out similarly as described above (example 1). In this case, a fluorescently-labeled reverse primer is used in the reaction mixture at a concentration exceeding the concentration of the forward primer by 5-10 times. Amplification is carried out in accordance with the instructions for the set of reagents.

15 μl of hybridization buffer (4 ^x SSC solution, 0.1% SDS) and 15 μl of the aqueous phase of the PCR mixture obtained as a result of amPCR are added to the required number of microcentrifuge tubes with a microdoserter and mixed for 20-30 s on a vortex with a frequency rotation of at least 1500 min ^-1 to obtain a hybridization mixture. DNA denaturation was carried out for 5 min at 94 ° C, followed by cooling on ice. 30 μl of a hybridization mixture are taken from each microcentrifuge tube with a microdoser and the whole mixture is introduced into the cell of the incubation chamber on the surface of the biological microchip with specific probes immobilized, the openings of the incubation chamber are sealed with a microplate film. Hybridization is carried out in a thermostat at a temperature of 37 ° C for 18 hours. After hybridization is complete, the incubation chamber is removed, each biological microchip is washed three times with distilled water at a temperature of 25 ° C and dried by centrifugation at 1000 min ^-1 5 min.

The hybridization picture is visualized on a biological microchip using a fluorescence scanner for biochips. The judgment on the belonging of the analyzed strain to a specific subspecies of the causative agent of tularemia is made on the basis of registration of hybridization with the corresponding probe (s). In this case, the hybridization picture of the analyzed strain and reference strains or control preparations of genomic DNA from them should correspond to the picture within the subspecies to which they belong (specificity 100%).

Example 6. Identification of strains of the causative agent of tularemia using oligonucleotide biochips.

Reference strains of F.tularensis or a set of genomic DNA preparations are used as a positive control for the identification of tularemia pathogen strains on oligonucleotide biochips.

All stages of the analysis on oligonucleotide biochips are carried out as described above (example 4). The hybridization picture of the analyzed sample is compared with the hybridization picture of the reference strains or genomic DNA preparations and the affiliation of the analyzed strain to a specific subspecies of the tularemia pathogen is determined.

Example 7. Evaluation of internal quality control of a reagent kit for determining the affiliation of crops to the species Francisella tularensis and the genus Francisella by PCR in accordance with WHO recommendations.

Two sets of reagents with primers based on 16S rDNA (F5-F11) recommended by the WHO Tularemia Guide (6) were prepared for studies. To assess the sensitivity of PCR with these kits, we used bacterial suspensions of the reference strain F. tularensis subspecies tularensis of genetic group AI with a concentration of 1 × 10 ⁵ -1 × 10 ³ m.k./ ml Samples were prepared for disinfection in the same way as described in Example 1. DNA isolation, PCR, and analysis of the results were carried out in the same way as in Example 1. Agarose gel electrophoresis was performed as described in Example 2.

When conducting PCR with two sets of reagents with primers based on 16S rDNA on the electrophoregram (figure 3), the following fragments were recorded:

Sample 1. F.tularensis subspecies tularensis of the genetic group AI at a concentration of 1 × 10 ⁵ m.k. / ml (1st set) - a fragment of 1104 bp

Sample 2. F.tularensis subspecies tularensis of the genetic group AI at a concentration of 1 × 10 ⁴ m.k. / ml (1st set) - a fragment of 1104 bp

Sample 3. F.tularensis subspecies tularensis of the genetic group AI at a concentration of 1 × 10 ³ MK./ml (1st set) - the result is doubtful.

Sample 4. Negative control - no fragment.

Sample 5. F.tularensis subspecies tularensis of the genetic group AI at a concentration of 1 × 10 ⁵ m.k. / ml (2nd set) - a fragment of 1104 bp

Sample 6. F.tularensis subspecies tularensis of the genetic group AI at a concentration of 1 × 10 ⁴ m.k. / ml (2nd set) - a fragment of 1104 bp

Sample 7. F.tularensis subspecies tularensis of genetic group AI at a concentration of 1 × 10 ³ m.k. / ml (2nd set) - a fragment of 1104 bp

The results indicate that the sensitivity of PCR with a set of reagents with primers based on 16S rDNA No. 1 is 1 × 10 ⁴ MK./ml, with a set of reagents with primers based on 16S rDNA No. 2 is 1 × 10 ³ m. K. / ml. Based on this, for further diagnostic studies, it seems advisable to use a reagent kit with primers based on 16S rDNA No. 2.

Example 8. The assessment of the specificity of the sera diagnostic tularemia and preparations of immunoglobulins diagnostic tularemia.

The specificity of diagnostic tularemia sera and diagnostic tularemia immunoglobulin preparations are assessed using a complete collection of reference F.tularensis strains.

Strains of F.tularensis are grown on solid nutrient media (FT agar) at a temperature of 37 ° C for 48 hours. Colonies with typical morphology (in S-form), typical morphology of cells in smears and tinctorial properties are selected.

When setting up immunological reactions, preparation of F.tularensis strains and disinfection of cultures is carried out in accordance with SP 1.3.1285-03 (12).

A. To perform ELISA (enzyme-linked immunosorbent assay), 96 well plates are sensitized by suspension of microorganisms of the reference F.tularensis strains in a 0.9% sodium chloride solution at a concentration corresponding to 10 units. industry standard sample turbidity (OSO 42-28-85P), equivalent to a concentration of 5 · 10 ⁹ MK / ml F. tularensis, at a temperature of 37 ° C for 2 hours or at a temperature of 4 ° C for 18 hours. binding sites are blocked with a 0.5% solution of bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 30 min at 37 ° C. The wells of the plates are washed three times by the FSB. Then, the test tularemia serum or immunoglobulins are added to the wells in working dilution in the FSB and incubated at 37 ° C for 1 h. After washing the wells of the FSB plates three times, the solution of the antispecific conjugate in the FSB with tween-20 is added to a final concentration of 10% labeled with horseradish peroxidase in working dilution. Incubation is carried out at a temperature of 37 ° C for 1 h. After washing the FSB wells six times, a solution of a chromogenic substrate is introduced into the wells and incubated for 30 minutes at room temperature with constant shaking. The reaction is considered positive if the optical density in the experimental wells is 2 times higher than in the negative control. In the wells of the negative control, FSB is added instead of antibodies.

The specificity of serum and immunoglobulins is determined by the formula:

C = (PI 100%) / (PI + LO), where

C - specificity

PI - truly positive results,

LO - false negative results.

B. To determine the specificity of tularemia sera and immunoglobulins in dot immunoassay, a nitrocellulose membrane with a pore diameter of 0.22 μm, divided into squares of 5 × 5 mm, is used as a substrate for sensitization with suspension of microorganisms. In each square, 2 μl of a suspension of microorganisms of the reference F.tularensis strains prepared as described in item A. Samples are fixed at 11 ° C for 10 minutes. Subsequent analysis steps are carried out in accordance with paragraph A. Water-insoluble substrates (O-dianisidine, diaminobenzidine, etc.) are used as a chromogen.

Example 9. Determination of the sensitivity of diagnostic sera of tularemia and preparations of immunoglobulins of diagnostic tularemia.

The sensitivity assessment of tularemia sera and immunoglobulins is performed using a complete collection of reference F.tularensis strains or individual collection strains.

A. For ELISA, 96-well plates with diagnostic tularemia serum at a concentration of 10-30 μg / ml at a temperature of 37 ° C for 2 hours or at a temperature of 4 ° C for 18 hours are sensitized. Free binding sites are blocked with a 0.5% BSA solution in the FSB for 30 min at a temperature of 37 ° C. The wells of the plates are washed three times by the FSB. Then, suspensions of the microorganisms of the reference F.tularensis strains in a 0.9% sodium chloride solution at a concentration corresponding to 10 units are added to the wells. industry standard sample turbidity (OSO 42-28-85P), equivalent concentration of 5 × 10 ⁹ MK./ml F.tularensis, and titrated to a final concentration of 1.5 × 10 ⁴ MK./ml (in 16 well ) The plates are incubated at 37 ° C for 1 hour. Then, the test tularemia serum or immunoglobulins are added to the wells in working dilution in the FSB and incubated at 37 ° C for 1 hour. After washing the wells of the FSB plates three times, the solution of the anti-conjugate is added to the wells in the FSB with the addition of tween-20 to a final concentration of 10% labeled with horseradish peroxidase in working dilution. Incubation is carried out at a temperature of 37 ° C for 1 h. After washing the FSB wells six times, a solution of a chromogenic substrate is introduced into the wells and incubated for 30 minutes at room temperature with constant shaking. The reaction is considered positive if the optical density in the experimental wells is 2 times higher than in the negative control. In the wells of the negative control, FSB is added instead of antibodies. The sensitivity of antibodies is equal to the last dilution of a suspension of microorganisms with a positive reaction.

B. To determine the sensitivity of tularemia sera and antibodies in dot immunoassay, a nitrocellulose membrane with a pore diameter of 0.22 μm, cut into squares of 5 × 5 mm, is used as a substrate for sensitization with commercial immunoglobulins. 2 μl of commercial immunoglobulins at a concentration of 10-30 μg / ml are added to each square. Samples are fixed at 110 ° C for 10 minutes. Subsequent analysis steps are carried out in accordance with paragraph A.

Water-insoluble substrates (O-dianisidine, diaminobenzidine, etc.) are used as a chromogen (table).

Example 10. Determination of the activity of sera diagnostic tularemia and preparations of immunoglobulins diagnostic tularemia.

The sensitivity assessment of tularemia sera and immunoglobulins is performed using a complete collection of reference F.tularensis strains or individual collection strains.

A. For ELISA, 96-well plates are suspended by suspension of reference F.tularensis strains in 0.9% sodium chloride solution at a concentration of 10 units. industry standard sample turbidity (OSO 42-28-85P), equivalent concentration of 5 × 10 ⁹ MK / ml F.tularensis at a temperature of 37 ° C for 2 hours or at a temperature of 4 ° C for 18 hours. binding block 0.5% solution of BSA in the FSB for 30 min at a temperature of 37 ° C. The wells of the plates are washed three times by the FSB. Then, test tularemia serum or immunoglobulins in a 1:10 dilution are added to the wells and titrated to a final dilution of 1: 327680 (in 16 wells). The plates are incubated at 37 ° C for 1 h. After washing the wells of the FSB plates three times, a solution of the antispecific conjugate in the FSB with tween-20 is added to the wells to a final concentration of 10% labeled with horseradish peroxidase in working dilution. Incubation is carried out at a temperature of 37 ° C for 1 h. After washing the FSB wells six times, a solution of a chromogenic substrate is introduced into the wells and incubated for 30 minutes at room temperature with constant shaking. The reaction is considered positive if the optical density in the experimental wells is 2 times higher than in the negative control. In the wells of the negative control, FSB is added instead of immunoglobulins. The activity of the test serum (immunoglobulins) is equal to the last dilution with a positive reaction.

B. To determine the activity of tularemia sera and immunoglobulins in dot immunoassay, a nitrocellulose membrane with a pore diameter of 0.22 μm, divided into squares of 5 × 5 mm, is used as a substrate for sensitization with suspension of microorganisms. 2 μl of a suspension of microorganisms of the reference F.tularensis strains is added to each square. Samples are fixed at 110 ° C for 10 minutes. Subsequent analysis steps are carried out in accordance with paragraph A. Water-insoluble substrates (O-dianisidine, diaminobenzidine, etc.) are used as a chromogen.

Information sources

1. Garrity G. M., editor. Bergey's Manual of Systematic Bacteriology: Springer New York; 2005. Vol. 2. 500 p.

2. Guidelines "Epidemiological surveillance of tularemia" MU 3.1.2007-05.

3. Patent SU, 1839960, A1, A61K 39/02, C12N 1/21.

4. Ikaheimo I., Syrjala H., Karhukorpi J. et al. In vitro antibiotic susceptibility of Francisella tularensis isolated from animals and animals // J. Antimicrobial Chemotherapy. - 2000. - Vol. 46. - P.287-290.

5. Nübel U., Reissbrodt R., Weller A. et. al. Population structure of Francisella tularensis // J. Bacteriol. - 2006. - Vol. 188. - P.5319-5324.

6. WHO Guidelines on Tularaemia // WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland. - 2007. - 115 p.

7. Product catalog of NPO SibEnzyme 2009 (http://www.sibenzyme.ru).

8. Vircell Microbiologists 2010 product catalog (www.biochemmack.ru).

9. “Organization of the work of laboratories using nucleic acid amplification methods when working with material containing microorganisms of pathogenicity groups I-IV”, MU 1.3.2569-09.

10. Forsman M., Sandstrom G., Sjostedt Analysis of 16S ribosomal DNA sequences of Francisella strains and utilization for determination of the phylogeny of the genus and for identification of strans by PCR // International Journal of Systematic Bacteriology / - 1994. - Vol. .44. - P.38-46.

11. Broekhuijsen M., Larsson P., Johansson A. et al. Genome-wide DNA microarray analysis of Francisella tularensis strains demonstrates extensive genetic conservation within the species but identifies regions that are unique to the highly virulent F. tularensis subsp.tularensis // J. Clin. Microbiol. - 2003. - Vol.41. - P.2924-2931.

12. Sanitary and epidemiological rules “Safety of work with microorganisms of I-II pathogenicity (danger) groups” SP 1.3.1285-03.

A collection of strains of the microorganism Francisella tularensis of various subspecies for genetic and immunological studies and a set of control preparations from them for genetic studies No. p / p Strain F. tularensis Sensitivity of diagnostic tularemia serum, (m.k. / ml) The sensitivity of tularemia immunoglobulins, (m.k. / ml) IFA * DIA * IFA * DIA * one KM 3 5 · 10 ⁵ 1 · 10 ⁵ 2.510 ⁶ 1 · 10 ⁵ 2 KM 5 1 · 10 ⁶ 1 · 10 ⁶ 2.510 ⁶ 1 · 10 ⁶ 3 KM 6 1 · 10 ⁵ 1 · 10 ⁶ 5 · 10 ⁵ 1 · 10 ⁶ four KM 4 1 · 10 ⁵ 1 · 10 ⁵ 1 · 10 ⁶ 1 · 10 ⁶ * ELISA - in enzyme-linked immunosorbent assay, DIA - in dot-immunoassay

Claims (4)

1. A set of bacteria strains of the species Francisella tularensis to obtain a set of control DNA preparations used for genetic diagnostic studies, containing bacteria strains of Francisella tularensis subspecies tularensis genetic group AI, KM 7 and genetic group AII, KM6, subspecies mediasiatica, KM 4, subspecies holarctica biovar japonica, KM 5, biovar Ery ^s , KM 3, biovar Ery ^R , KM 8, deposited in the State collection of pathogenic bacteria "Microbe". 2. A set of control DNA preparations for genetic diagnostic studies in determining the type, subspecies and biovar of the causative agent of tularemia, containing genomic DNA or combinations of genomic DNA of bacterial strains of the species Francisella tularensis, presented in the kit according to claim 1. 3. The kit according to claim 2, characterized in that it contains a combination of genomic DNA of strains of Francisella tularensis subspecies tularensis of the genetic group AI, KM 7 and the genetic group of AN, KM 6 in equal proportions. 4. The kit according to claim 2, characterized in that it contains a combination of genomic DNA of strains of Francisella tularensis subspecies holarctica biovar japonica, KM 5, biovar Ery ^s , KM 3, biovar Ery ^R , KM 8 in equal proportions.

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