Method of differentiating bacillus anthracis from other closely related species of genus bacillus based on determining differences in structure of chromosomal genes

FIELD: biotechnology.

SUBSTANCE: method includes sample preparation, DNA isolation, PCR statement. At that in carrying out PCR, the oligonucleotide primers are used, which are complementary to sequences of the chromosomal genes fliC and hom2, having the following sequences: fliC-F: 5'-TGGAGCAGTAACAATTGG-3', fliC-R: 5'-GCACCACTGATAGAAATGTTAG-3', hom2-F: 5'-GACGTGTTAAAAGAAGCCCA-3', hom2-R: 5'-CACCAATTTCGTCTTTTACA-3', followed by electrophoretic analysis of the amplification products, when the formation of the amplification product is 153 bps in size it is indicative of belonging of the strain under study to the species B.anthracis, formation of the amplification product with size of 550 bps is indicative of belonging of the strain under study to the other species of the genus Bacillus.

EFFECT: invention enables to differentiate effectively the bacillus anthracis from other species of bacilli.

1 dwg, 1 ex

 

The invention relates to the field of molecular Microbiology, in particular for the diagnosis of anthrax. The proposed invention allows to quickly and efficiently identify DNA of Bacillus anthracis in a biological material and the environment when conducting diagnostic tests in practical human and animal health and to implement a reliable differentiation from other bacillary species.

The causative agent of anthrax - gram-positive spore-forming microorganism belonging to the genus Bacillus. In the closest phylogenetic relationship with Century anthracis are the species B. cereus, B. thuringiensis, B. mycoides, B. pseudomycoides and B. weihenstephanensis. The similarity of genetic, morphological and physiological characteristics allows to allocate them to a particular taxonomic group, the so-called "1-St group of bacilli".

The classic design of the study material for the presence of anthrax based on the determination of the phenotypic characteristics colony morphology, sensitivity to penicillin in the test "pearl necklaces", sensitivity to diagnostic bacteriophages, ability to the formation of L-glutamine capsules on special media, the absence of hemolysis on agar with defibrinating the blood of the lamb, the lack of mobility and activity of alkaline phosphatase. However, quite often from Aut strains Century anthracis that one or more characteristics different from the typical characteristics.

The known method of differentiation of strains of Bacillus anthracis virulence was demonstrated in vitro, performed on special nutrient media for testing the formation of capsules and anthrax toxin in the atmosphere of CO2[1]. The known method of identification Century anthracis with the differentiation of strains for the production of capsules, Pro-tektivnye antigen and antigen S-layer, consisting of the seed culture of the strain on a special testing environment for the formation of capsules in an atmosphere of CO2and the subsequent formulation of the reaction immunodiffusion using sera to protective antigen and protein S-layer of Bacillus anthracis [2]. However, these methods are designed to work with a pure culture of C. anthracis, with their help it is impossible to carry out the detection of anthrax in biological material and environmental objects and differentiation from other members of the genus Bacillus.

Modern development of genetics and molecular Microbiology allows the diagnostics of pathogenic bacteria based amplication and sequencing technologies. Molecular genetic differentiation has high resolution and eliminates various interpretations associated with phenotypic variable is STU bacteria. In the genome of the causative agent of anthrax, in addition to the chromosome, there are two plasmid - pXO1 and pXO2. Plasmid genes determine a synthesis of the main factors of pathogenicity of Bacillus anthracis. The presence of plasmids pXO1 and pXO2 a differential diagnostic feature that distinguishes a Century anthracis from other members of the genus Bacillus. Almost all domestic and foreign PCR-display system of anthrax is based on the identification of sequences of plasmid genes.

There is a method of detecting anthrax using a commercial kit Gensim - Test-system for detection of DNA Century anthracis Rho+by polymerase chain reaction" (FCUS antiplague research Institute "Microbe", Saratov) [3]. The kit contains primers, complementary to the fragment sequences pagA gene, located on the pXOl plasmid. However, this test system will not detect monopedigree strains Century anthracis containing only plasmid pXO2, or besplatnye derivatives. In addition, it is impossible to distinguish vaccine strains Century anthracis from virulent.

There is a method of detecting anthrax using a commercial kit "Amplisense®Century anthracis - FRT: reagent kit for detecting a DNA of vegetative forms and spores of Bacillus anthracis in a biological material and the environment method polimeraznoi reaction (PCR) with hybridization-fluorescence detection in "real time" (fsri Central research Institute of epidemiology, Moscow) [4]. The test system is based on the identification of the fragment of the pagA gene, localized in the structure of the plasmids pXO1 and Sara gene located on the plasmid pXO2. However, it does not detect strains of Bacillus anthracis lacking the plasmid. Bacterial plasmids are less stable genetic elements than the chromosomal DNA, and can have different frequency to eliminirovat. On the other hand, the plasmid is identical to pXO1 and pXO2, found in certain strains of the closely related C. anthracis bacilli species [5, 6, 7]. Some of these unusual strains isolated from humans or animals with symptoms resembling anthrax.

Greater specificity have a test system containing primers for fragments of the chromosomal genes. However, the search for species-specific chromosomal loci is difficult due to the high homology of the chromosomal DNA of the anthrax and individual members of the species B. cereus [8].

Known methods of detection Century anthracis and intraspecific differentiation of plasmid composition, based on the integrated use of primers for the gene fragments pag, cya, lef, localized on plasmid pXO1, cap gene located on the plasmid pXO2 and chromosomal sequence V [9, 10]. However, the uniqueness of the token VA for anthrax questioned after detection of individual strains Century ceeus and B. thuringiensis containing a sequence A, identical with the causative agent of anthrax [11].

A known method for the identification and differentiation of C. anthracis using multiplex amplication test systems, including primers for fragments of plasmid genes cya and SARS, as well as chromosomal sap gene encoding protein S-layer [12]. However, according to the genome-wide sequences represented in the databases of the National Center for Biotechnology Information (NCBI), the nucleotide sequence of the gene sap strains Century anthracis and certain other species of the genus Bacillus are identical.

Qi Y. et al., comparing chromosomal sequence bacillary strains found in Bacillus anthracis synonymous replacement of one nucleotide in the rpoB gene encoding the β-subunit of RNA polymerase [13]. The prospects of the use of this marker for the implementation of differentiation Century anthracis among phylogenetically related species of the genus Bacillus. However, in subsequent testing 319 bacillary strains was identified strain of B. cereus with rpoB gene sequence that is identical to a similar sequence in B. anthracis [14].

Agaisse H. et al. in the process of comparative analysis of sequenced genomes Century anthracis and B. cereus showed polymorphism of a single nucleotide in the chromosomal gene pleiotropic regulator plcR [15]. Transcription the ion regulator plcR responsible for different functions of cells, related to the pathogenicity of the microorganism and survival in a hostile environment. A nonsense mutation in the plcR gene is used in the typing of strains Century anthracis different origin and refers to the canonical SNP (single nucleotide polymorphism) [16]. Hurtle W. et al. revealed a single synonymous nucleotide substitution in the chromosomal gyrA gene of strains Century anthracis, which distinguish them from members of other bacillary species [17]. On the basis of the gyrA gene polymorphism was created probe with a fluorescent label. However, for the detection of genetic changes in the form of single nucleotide substitutions need special equipment to perform real-time PCR or sequencing. In addition, mutations in the form of single nucleotide substitutions prone reverse reversion.

Irenge L. et al. was proposed two-step real-time PCR for the diagnosis of anthrax infection [18]. At the first stage of screening strains Century anthracis using plasmid genes of Bacillus anthracis, the second stage is a more specific identification of strains Century anthracis using chromosomal target genes pur and ptsI. However, the diagnostic study carried out in two stages, more time and less economical.

Wielinga, P. et al. developed multiloci real-time PCR for simultaneous detection chromoso the nogo and plasmid loci anthrax. As a chromosomal marker selected lambda propag 3 type, which is part of the conservative region of DNA Century anthracis [19]. However, there is a possibility of recombination events in the genome of C. anthracis, leading to the elimination of propaga.

In 2011, Ahmod N. et al. found a deletion 72 BP in a conservative region of a chromosome anthrax, encoding ATP synthase gene WAAS [20]. Testing with specific primers 174 bacillary strains, including 39 strains of anthrax, showed the presence of mutations in the vast majority of strains Century anthracis, except Century anthracis A. Chromosomal marker weas was recommended for the differentiation of Bacillus anthracis from bacillary strains of other species by PCR and persecutione. However, the use of chromosomal mutations in the form of deletion of part of the nucleotides in any gene can be used to detect the microorganism. For simultaneous indication of a bacterial pathogen and its differentiation from closely related species optimally using multiple species-specific loci.

The objective of the invention is to develop a method that allows detection of the causative agent of anthrax during the diagnostic studies and conduct differentiation from other members of the genus Bacillus.

The technical result of the conclusion is to be effective in indicating Bacillus anthracis and improving the reliability of its differentiation from strains phylogenetically related species.

The technical result is achieved by the display of anthrax by polymerase chain reaction with primers on chromosome genes, which includes sample preparation, DNA isolation, formulation PCR using synthetic oligonucleotide primers complementary to the sequences of chromosomal genes fliC and hom2.

The primers for these genes have the following sequence:

fliC-F: 5'-TGGAGCAGTAACAATTGG-3',

fliC-R: 5*-GCACCACTGATAGAAATGTTAG-3',

hom2-F: 5'-GACGTGTTAAAAGAAGCCCA-3',

hom2-R: 5'-CACCAATTTCGTCTTTTACA-3*.

The results were evaluated according to the presence of amplified gene fragments and their sizes. The formation of the product of amplification with primers for a fragment of the gene fliC size 153 BP indicates the affiliation of strain to the species V. anthracis. The presence of the amplification product with primers for a fragment of the gene hom2 a 550-BP, in contrast, eliminates the assignment to isolate the causative agent of anthrax.

The inventive method is based on using as a DNA target fragments of the chromosomal genes, fliC and hom2. Earlier in the process of comparative analysis of nucleotide sequences of genes livelihoods bacillary strains we have identified specific to anthrax chromosomal mutations. One of the high-affinity sites was found in the gene fliC determining the synthesis of flagellin - chief whom Ananta bacterial flagellum. At position 383 BP from the beginning of the fliC gene in strains Century anthracis is present insert 231 nucleotides, leading to the cessation of the synthesis of flagellin. When setting PCR with primers complementary to the fragment sequences of the inserts, the formation of the product of amplification occurs in the interaction with DNA Century anthracis.

Additional DNA target for diagnostic studies is that we discovered a mutation in a gene hom2, encoding the enzyme homoerythromycin. The deletion of 42 nucleotides at position 1042 BP on 1083 BP from the beginning of the gene hom2 blocks the synthesis of L-homoserine, from which a series of sequential reactions are formed methionine, threonine and cysteine. Given the relatively small extent of the defect DNA primers for gene hom2 selected so that the sequence of one of them had directly to the region of the deletion. In this case, the formation of amplification product is detected only in reactions with DNA strains bacillary species other than C. anthracis. The use of additional chromosomal locus increases the reliability differentiation anthrax from strains phylogenetically related species. Mutation in the gene hom2, apparently, occurred at the initial stage of branching from a common precursor and formation of the Century anthracis as a separate species. This is the case it is species-specific genetic marker.

The possibilities of the proposed method provide for the introduction of plasmid markers, which allows simultaneously with the detection of Bacillus anthracis to determine the facilities to isolate virulent vaccine or atypical strains. Calculation of primers and selection conditions of polymerase chain reaction carried out with a view to future use in the same reaction mixture of primers for fragments of chromosomal genes fliC, hom2 and plasmid determinants pagA, cap And [19]. Size formed in multiloci PCR amplification products allows them to be distinguished from each other in an electrophoretic separation in agarose gel. Staging a one-step PCR economically it is more efficient and requires less time for research.

The method is as follows:

The isolation of DNA from material previously decontaminated according to the standard procedure carried out in accordance with MU 1.3. 2569-09 "Organization of work of the laboratories using methods of nucleic acid amplification when working with material containing microorganisms 1 - IV groups of pathogenicity (M, 2009).

Polymerase chain reaction carried out according to standard methods (MU 1.3. 2569-09) using the above primers for genes hom2 and fliC.

Oligonucleotide primers used for PCR amplification of gene fragments hom2 and fliC, calculated on the basis of the e nucleotide sequences of these genes from bacillary strains, represented in NCBI databases: B. anthracis Ames, Ames 0581, Steme, CDC 684, A0248; B. cereus ATCC14579, ATCC10987, ZK, AH187, B4264, AH820, G9842, Q1, 03BB102; Bacillus thuringiensis 97-27, Al Hakam, BMB171; B. weihenstephanensis KBAB4_4052, B. licheniformis ATCC 14580, DSM13; B. B. megaterium QM B1551, DSM 319; B. subtilis subsp.spizizenii, BSU35150, BSn5. Primers were analyzed using the computer program BLAST on a web server NCBI (http://www.ncbi.nlm.nih.gov/BLAST/) to confirm their specificity and lack of homology with the nucleotide sequences of bacillary strains of the species present in the databases (GenBank, EMBL, DDBJ).

Using designed primers is carried out in PCR amplification of gene fragments hom2 and JliC determine the presence and dimensions of the resulting amplicons. Primers for the genes hom2 and fliC have the following sequence:

fliC-F: 5'-TGGAGCAGTAACAATTGG-3',

fliC-R: 5'-GCACCACTGATAGAAATGTTAG-3',

hom2-F: 5'-GACGTGTTAAAAGAAGCCCA-3',

hom2-R: 5'-CACCAATTTCGTCTTTTACA-3'.

Polymerase chain reaction with amplification of the gene fragments hom2 and fliC carry out the following program: one cycle of 94°C for 5 min, 30 cycles at 94°C for 30 sec, 58°C 30 sec, 72°C for 30 s and a final cycle of 3 min at 72°C. Electrophoretic analysis of PCR products is carried out by electrophoresis in 2% agarose gel relative to the standard markers molecular masses ranging in size from 100 to 3000 BP

For strains of Bacillus anthracis PCR product corresponds to the size of 153 BP, for bacillary strains of other species - 550 BP

Still the way the developed method allows in a short period of time to detect the causative agent of anthrax, as well as conduct its reliable differentiation from other members of the genus Bacillus.

The invention is illustrated by example.

Example. Study material containing a culture of the strain Century anthracis or strain other bacillary species (Model experiment).

Disinfection of the investigated samples is conducted according to guidance MU 3.5.5.1034-01 "Disinfection of the studied material, the infected bacteria I-IV groups of pathogenicity, the PCR".

DNA isolation is performed with the help of a Set of reagents for sample preparation (DNA extraction)" manufactured FCUS antiplague research Institute "Microbe".

The reaction mixture for the production of a PCR reaction is prepared according to the recipe:

deionized water is 12.5 ál,

a solution of dNTP (2.5 mm) - 2 ál,

primer fliC-F (12 PM/ μl) and 0.5 μl,

primer fliC-R (12 PM/ μl) and 0.5 μl,

primer hom2-F (12 PM/ μl) and 0.5 μl,

primer hom2-R (12 PM/ μl) and 0.5 μl,

the reaction buffer NPF (×10) and 2.5 µl,

MgCl20.25 M to 0.8 ál,

the enzyme Taq polymerase (5 units/ ál) - 0.2 µl,

the sample DNA 5 ál.

The total reaction mixture of 25 μl of 1 sample.

Amplification of fliC gene fragments and hom2 in PCR performed according to the scheme: denaturation - 94°C for 5 min, 30 cycles at 94°C for 30 sec, annealing Primero is - 58°C - 30 sec, elongation of the chain 72°C for 30 s and a final cycle of 3 min at 72°C. Electrophoretic analysis of PCR products is carried out in a 2% agarose gel.

In all samples containing DNA Century anthracis, is synthesized amplification product size 153 gel that defines the insertion in the gene fliC. When DNA testing of representatives of various bacillary species register a product of amplification of a 550 BP, revealing an unmodified gene sequence hom2. Due to the deletion of part of the nucleotide sequence of the gene hom2 in strains of anthrax amplification with designed primers for fragment in these samples does not occur (drawing). The drawing shows the results of PCR analysis with primers for fragments of genes, fliC and hom2 strains Century anthracis and other bacillary species: 1. Century anthracis Pasteur; 2. Century anthracis 55; 3. Century anthracis Ihtiman; 4. Century anthracis CM (7); 5. Century anthracis CM (8); 6. Century anthracis Sterne 34F2; 7. Century anthracis STI-1; 8. Century anthracis CM; 9. Century anthracis 29; 10. Century anthracis KM 91; 11. the molecular mass marker' O ' GeneRuler™ 100bp DNA Ladder Plus redy-to-use" (Fermentas) (3000, 2000, 1500, 1200, 1000, 900, 800, 700, 600, 500,400, 300, 200, 100 P.N.); 12. Century thuringiensis HD-1; 13. B. thuringiensis 2090; 14. B. thuringiensis 69/6; 15. B. cereus 569; 16. B. cereus 108; 17. B. cereus 8; 18. B. pseudoanthracis 104.

The sensitivity of the amplification reaction with primers fliC-F, fliC-R, hom2-F and hom2-R were evaluated in the study samples DNA extracted from ten-fold dilutions of pure culture of the pathogen Siberian language is s Century anthracis, and amounted to 1×102-1×104spores/ml.

Literature

1. Eremenko H., Buravceva N.P., Funtikova so-CALLED. The method of differentiation of strains of Bacillus anthracis virulence was demonstrated in vitro // Patent RF №2101351 (C12N 1/20, C12Q 1/04, C12N 1/20, C12R 1:07) - published 10.01.1998,

2. Barkov I.A., Barkov A.M., Alekseev V.V., ALEXANDER Lipnitsky a Method for the identification of Bacillus anthracis with the differentiation of strains for the production of capsules, protective antigen and antigen S-layer // Patent RF №2376385 (C12Q 1/04, C12R 1/07, G01N 33/559) - published 20.12.2009,

3. Tuchkov IV Design and implementation of genetic diagnostic test system for detecting the DNA of the anthrax // abstract. dis.... Kida. the honey. Sciences. - Saratov, 2005. - 19 S.

4. Abdrashitova A.S., sapina L.V., Malahaeva A.N., Aspen N.A. Study of the effectiveness of the kits for the detection of DNA vozbuditelei anthrax, Brucellosis, cholera PCR in "real time" // Modern technologies in improving prevention and response to emergencies in public health sanitary-epidemiological nature (proceedings of XI International scientific-practical conference). Saratov. - 2012. - S-15.

5. Avashia, S., Riggins W., Lindley, A. Hoffmaster, Drumgoole R., Nekomoto,T., Jackson, P., Hill, K., K. Williams, L. Lehman, M. Libal, P. Wilkins, Alexander J., Tvaryanas, A., T. Betz Fatal pneumonia among metalworkers due to inhalation exposure t Bacillus cereus containing Bacillus anthracis toxin genes // Clin. Infect. Dis. - 2007. - V.44. - P.414-416.

6. Hoffmaster, A., Ravel J., Rasko, D., Chapman G., M. Chute, Marston C, De C., Sacchi C, Fitzgerald C, Mayer L., M. Maiden, Priest F., Barker M., Jiang L., Cer R, Rilstone j, Peterson S., R. Weyant, Galloway D., Read, T., Popovic T., Fraser C. Identification of anthraxtoxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax // Proc. Natl. Acad. Sci. USA - 2004. - V.101. - P.8449-8454.

7. Klee, S., Ozel M., Appel C., Boesch C, Ellerbrok, H., Jacob D., Holland G., F. Leendertz, Pauli G., Grunow R., Nattermann, H. Characterization of Bacillus anthracis-k& bacteria isolated from wild great apes from Cote dTvoire and Cameroon // J. Bacteriol. - 2006. -V.188.-P.5333-5344.

8. Tourasse, N., E. Helgason, A. Okstad, Hegna I., Kolsto, A. The Bacillus cereus group: novel aspects of population structure and genome dynamics // J. Appl. Environ. - 2006. -V.101.-P.579-593.

9. Patra G., Sylvestre, P., Ramisse V, Therasse J., Guesdon J. Isolation of a specific chromosomic DNA sequence of Bacillus anthracis and its possible use in diagnosis // FEMS Immunol. Med. Environ. - 1996. - V.15. - P.223-231.

10. Ramisse, V., Patra, G., Garrigue H., Guesdon J., Mock M. Identification and characterization of Bacillus anthracis bymultiplex PCR analysis of sequences on plasmids pXOl and Rho and chromosomal DNA // FEMS Environ. Letters. - 1996. - V.145. - P.9-16.

11. Patra G., Vaissaire J., Weber-Levy, M., Le Doujet C, Mock M. Molecular characterization of Bacillus strains involved in outbreaks of anthrax in France in 1997 // J. Clin. Environ. - 1998. - V.36. - P.3412-3414.

12. Tsygankova I. Eremenko, H., Ryazanov, A.G., E. Tsygankova Multiplex amplication test system for the identification and differentiation of Bacillus anthracis II). microbiol. - 2005. No. 3. - P.69-74.

13. Qi Y., Patra, G., Liang X., Williams L., Rose, S., Redkar, R., DelVecchio V. Utilization of the rpoB gene as a specific chromosomal marker for real-time PCR detection of Bacillus anthracis II Appl. Environ. Environ. - 2001. - V.67(8). - P.3720-3727.

14. Zasada A., Gierczynski R., Raddadi n, Daffonchio D., Jagielski M. Some Bacilus thuringiensis strains share rpoB nucleotide polymorphisms also present in Bacillus anthracis II J. Clin. Environ. - 2006. - V.44. - P.1606-1607.

15. Agaisse, H., Gominet, M., A. Okstad, Kolstø, A., D. Lereclus PlcR is a pleiotropic regulator of increasing interest among virulence factor gene expression in Bacillus thuringiensis // Environ Mol. - 1999. - V.32. - P.1043-1053.

16. Easterday R., Van Ert, M., Simonson T., Wagner D., Kenefic L., Allender C, Keim P. Use of single nucleotide polymorphisms in the plcR gene for specific identification of Bacillus anthracis II J. Clin. Environ. - 2005. - V.43 (4). - P.1995-1997.

17. Hurtle W., Bode, E., Kulesh D., Kaplan R., Garrison J., Bridge, D., House, M., Frye, M., Loveless Century, Norwood D. Detection of the Bacillus anthracis gyrA gene by using a minor groove binder probe. J. Clin. Environ. - 2004. - V.42. - P.179-185.

18. Irenge L., Durant J., H. Tomaso, Pilo, P. J. Olsen, Ramisse V, Mahillon J., Gala J. Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples, Appl. Environ. Biotechnol. - 2010. - V.88.-P.1179-1192.

19. Wielinga, P., Hamidjaja R., Agren J., Knutsson, R., Segerman Century, Flicker M., Ehling-Schulz M., Groot, A., Burton J., Brooks T., Janse I., Rotterdam B. A multiplex real-time PCR for identifying and differentiating B. anthracis virulent types // International J. Food Environ. - 2011. - V. 145. - P. 137-144.

20. Ahmod N., Gupta R., Shah H. Identification of a Bacillus anthracis specific indel in the yeaC gene and development of a rapid pyrosequencing assay for distinguishing B. anthracis from the B. cereus group // J. Environ. Methods. - 2011. - V. 87. - P. 278 - 285.

The method of differentiation of anthrax from other closely related species of the genus Bacillus based on determining the differences in the structure of the chromosomal gene, involving sample preparation, DNA isolation, PCR staging, characterized in that when PCR is used oligonucleotide primers complementary to the sequences of chromosomal genes fliC and hom2 with the following paragraph is coherence:
fliC-F: 5'-TGGAGCAGTAACAATTGG-3',
fliC-R: 5'-GCACCACTGATAGAAATGTTAG-3',
hom2-F: 5'-GACGTGTTAAAAGAAGCCCA-3',
hom2-R: 5'-CACCAATTTCGTCTTTTACA-3'
with subsequent electrophoretic analysis of amplification products, in which the formation of amplification product size 153 BP indicates the belonging of the examined strains to the species V. anthracis, the formation of the product of amplification of a 550 BP indicates the belonging of the examined strain to another species of the genus Bacillus.



 

Same patents:

FIELD: biotechnology.

SUBSTANCE: strain of fungus Aspergillus oryzae Amy T-52-3-21 produces maltogenic α-amylase, and it is deposited in the All-Russian Collection of Microorganisms Institute of Biochemistry and Physiology of Microorganisms n.a. GK Scriabin RAS under the number F-4476D. The strain is made on the basis of strain Aspergillus oryzae of All-Russian Collection of Microorganisms F-3927D using the genetic engineering methods. Activity of α-amylase at 120 h of growth of the strain is 600-640 units/ml.

EFFECT: higher level of activity of maltogenic α-amylase.

1 tbl, 2 ex

FIELD: biotechnologies.

SUBSTANCE: strain of bacteria Bacillus vallismortis VKPM V-11017 is proposed - destructor of oil and oil products. Strain may within short period of time in the wide range of temperatures from +8 to +37°C degrade oil by 78.3%.

EFFECT: strain may be used to clean soil and water contaminated by oil and oil products, such as diesel fuel, motor oil, gas condensate.

3 tbl, 5 ex

FIELD: biotechnologies.

SUBSTANCE: strain of bacteria Bacillus atrophaeus VKPM V-10592 is grown, and suspension is prepared from it, which is introduced into permafrost soil and water environment. Maintained at the specified parameters from 7 to 60 days, and then they determine quantity content of oil and oil products in permafrost soil and water environment.

EFFECT: invention makes it possible to reduce time for oil and oil product denaturation and to reduce their concentration in permafrost soil and water environment.

5 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Claimed is method of obtaining androst-4,9(11)-dien-3,17-dione from phytosterol. Microbiological oxidative elimination of side chain at atom C17 with formation of 9α-hydroxyandrost-4-en-3,17-dione is performed. Biomass is separated. 9α-hydroxyandrost-4-en-3,17-dione is extracted from clarified cultural liquid with aprotic organic solvent, selected from aromatic hydrocarbons or organochlorine hydrocarbons. After that, reaction of 9α-hydroxygroup of 9α- hydroxyandrost-4-en-3,17-dione dehydration is carried out in obtained extract. As dehydration agent applied is mineral acid, which contains water and is selected from group, which includes orthophosphoric, pyrophosphoric and chloric acids. Mineral acid is applied in quantity from 1 to 10 mol per 1 mol of 9α- hydroxyandrost-4-en-3,17-dione. In the process of dehydration reaction removal of excessive water is carried out either in presence of effective quantity of pyrophosphoric acid or by azeotropic distillation.

EFFECT: invention makes it possible to intensify dehydration process with application of smaller quantity of mineral acid and exclude side product formation.

11 cl, 1 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, microbiology, and concerns the recovery and identification of pseudotuberculosis and intestinal yersiniosis agents (Y. Pseudotuberculosis and Y. Enterocolytica). A nutrient medium contains microbiological agar (dry), lactose, glucose, urea, calcium chloride, 1% alcoholic phenol red, 1% alcoholic methylene blue and distilled water in specific proportions.

EFFECT: invention enables reducing the length of studies.

3 dwg, 2 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Claimed is strain of Fusarium sambucinum, deposited in VKPM collection under number F-1161. Claimed strain is producent of protein food biomass.

EFFECT: invention makes it possible to accumulate biomass with high protein content with higher quantity of valuable unsaturated fatty acids, complete in composition.

2 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Claimed is method of obtaining food fungal biomass with high protein content. Multicycle deep cultivation of Fusarium sambucinum All-Russian collection of industrial microorganisms F-1161 on liquid nutritional medium, containing sources of carbon, nitrogen, mineral salts, separation and drying of wet fungus biomass are carried out. Cultivation is performed at pH from 3.5 to 7.0 under conditions of air aeration from 0.5 to 2.0 l/l/min. Temperature mode in each cycle of fermentation is supported from the beginning of the cycle to the point of switch at the level from 26 to 30°C, and further to the end of the cycle at the level from 22 to 25°C. Point of switch is determined by accumulation of biomass to concentration from 45 to 60% from maximally achievable in fermentation apparatus, or point of switch is determined by concentration of dissolved oxygen by its reduction to the value from 20 to 40% of saturation ( calculated per atmospheric air pressure).

EFFECT: invention makes it possible to obtain the largest accumulation of biomass with high protein content with specified quantity of nucleic acids.

4 ex

FIELD: biotechnology.

SUBSTANCE: growing of Staphylococcus aureus is carried out in a nutrient medium containing yolk-salt agar. At a stage of preparation for analysis the growth stimulators of Staphylococcus aureus are introduced into the nutrient medium in the form of aqueous solutions at concentrations of 10-4-10-6 wt %. The following compounds are used as growth stimulators: tris(2-hydroxyethyl)ammonium 4-chlorophenyl-sulfanylacetate or tris(2-hydroxyethyl)ammonium 2-chlorophenyloxyacetate or tris(2-hydroxyethyl)ammonium 2-methyl-4-chlorophenyloxyacetate or tris(2-hydroxyethyl)ammonium 1-benzylindol-3-yl-sulfanylacetate.

EFFECT: invention enables to accelerate growing of Staphylococcus aureus for diagnostics of infections, by reducing the time of growing from 48 to 6-9 hours in comparison with the control in a standard nutrient medium.

2 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and deals with recombinant strain of E. coli TG1(pRVMoscow3253G-L) for obtaining PCR-standards for quantitative determination of cDNA of rabies virus of strain "Moskva 3253".Recombinant strain is created on the basis of strain of E. coli TG1 by transformation with plasmid pRVMoscow3253G-L. Plasmid is obtained by ligation of fragment G-L of the region of genome of fixed rabies virus of strain "Moskva 3253", which has sequence SEQ ID NO1, into plasmid pGem-T. Also claimed is set of PCR-standards for quantitative determination of cDNA of rabies virus of strain "Moskva 3253" in rabies antigen. Set contains solutions of plasmid pRVMoscow3253G-L DNA in concentrations 108, 107, 105, 103 GE/ml. Concentration is determined by method of polymerase chain reaction, with hybridisation-fluorescence account of results.

EFFECT: invention contributes to standardisation of stage of preparation of rabies antigen in production of heterological anti-rabies immunoglobulin.

2 cl, 3 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry and molecular biology. Conservation of cells of Escherichia coli in presence of buffered 80-90% glycerol is performed. Cell envelopes are removed with 3% triton X-100. Cell supramolecular structures are successively extracted with increasing concentrations of salts: 0.14 M (bacterioplasm), 0.35 M (loosely linked with cell residue), 2 M NaCl (strongly linked with cell residue), and 6 M guanidine hydrochloride with 0.1% β-mercaptoethanol (cell residue). Acid hydrolysis is carried out in said fractions. Anthrone method is carried out, with preliminary purification of anthrone preparation. Calibration graph is built and quantity of hexoses is determined by means of calculation formula.

EFFECT: invention makes it possible to determine quantity of hexoses in supramolecular structures of bacterial cell of Escherichia coli.

3 dwg, 3 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to chronobiology. A method for measuring a circadian cycle on the basis of time-series data by expression levels obtained by measuring the expression levels of two clock-genes in biological samples taken from a subject three times a day. The clock-genes have different phases of the circadian measuring cycle of the expression level.

EFFECT: method provides the high accuracy measurement of the biological rhythm and minimises the quantities of samplings from the subject.

4 cl, 4 dwg, 6 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology and deals with method of quantitative determination of fixed rabies virus strain "Moskva 3253". Method includes decontamination and separation of RNA from virus-containing material, carrying out reaction of reverse transcription and polymerase chain reaction with hybridization-fluorescence account of results in "real time" mode with application of specific primers RV5-5'-GTTGGGCACTGAAACTGCTA-3', RV6-5'-GAATCTCCGGGTTCAAGAGT-3' and probe RV7-5'-ROX-AATCCTCCTTGAACTCCATGCGACAGA-BHQ2. Quantitative assessment of virus is determined on the basis of registration of signal of analysed sample fluorescence and its comparison with signal of fluorescence of PCR-standards, which contain different quantities of DNA-targets. Claimed method makes it possible to determine quantitative content of virus in rabies antigen of organ-tissue and culture origin.

EFFECT: application of invention contributes to standardisation of stage of rabies antigen preparation in production of heterological anti-rabies immunoglobulin.

2 tbl, 3 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to genetic engineering, more specifically to analysing the disorders related to ovarian carcinoma, and may be used in medicine. The method involves determining the methylation status of CpG-dinucleotide in the genome in each sequence of a group of sequences SEQ ID NO:1-10 using a set of probes specific for the above sequences and able to be hybridised with the sequence along the full length. The above sequences are used as a part of a chip for detection, diagnosis and monitoring of the proliferative disorders related to ovarian cell proliferation, as well as for detection of a predisposition to the proliferative disorders, or treatment of the proliferative ovarian disorders.

EFFECT: invention enables identifying the proliferative disorders in ovarian cells and detecting a genetic predisposition to the above disorders.

10 cl, 3 dwg, 2 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to molecular biology and pharmacology. What is presented is a method for the prediction of the pharmaceutical effectiveness of adalimumab for treating rheumatoid arthritis, wherein the method involves measuring a level of at least one of ADAMTS4 iRNA and ADAMTS5 iRNA in a sample taken from a subject, and determining if adalimumab is effective for rheumatoid arthritis in a subject on the basis of a level of at least one of ADAMTS4 iRNA and ADAMTS5 iRNA considered as a value.

EFFECT: method may be used in medicine in treating rheumatoid arthritis.

3 cl, 2 dwg, 8 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology and deals with set, which includes oligodeoxyribonucleotide primers and fluorescently labelled probe for identification of DNA of adenovirus of serotypes 3, 4, 7, 14, 21 by method of hybridization-fluorescence polymerase chain reaction in real time mode. Claimed primers and probe have the following structure: external primer 5'→3' 5'-AATGTARTTGGGTCTGTTRGGCAT-3' internal primers 5'→3' 5'-CCCWTCGATGMTGCCCC-3' 5'-TCMACGGGYACRAAGCGCA-3' probe 5'→3' ROX-CCTGTCCGGCGATGTGCAT-BHQ2.

EFFECT: invention possesses higher homology to currently circulating adenoviruses and makes it possible to identify DNA of adenoviruses of serotypes 3, 4, 7, 14, 21.

3 dwg, 1 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and deals with recombinant strain of E. coli TG1(pRVMoscow3253G-L) for obtaining PCR-standards for quantitative determination of cDNA of rabies virus of strain "Moskva 3253".Recombinant strain is created on the basis of strain of E. coli TG1 by transformation with plasmid pRVMoscow3253G-L. Plasmid is obtained by ligation of fragment G-L of the region of genome of fixed rabies virus of strain "Moskva 3253", which has sequence SEQ ID NO1, into plasmid pGem-T. Also claimed is set of PCR-standards for quantitative determination of cDNA of rabies virus of strain "Moskva 3253" in rabies antigen. Set contains solutions of plasmid pRVMoscow3253G-L DNA in concentrations 108, 107, 105, 103 GE/ml. Concentration is determined by method of polymerase chain reaction, with hybridisation-fluorescence account of results.

EFFECT: invention contributes to standardisation of stage of preparation of rabies antigen in production of heterological anti-rabies immunoglobulin.

2 cl, 3 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biochemistry, in particular to set of synthetic oligonucleotides for identification of DNA of parodontopathogenic microorganism Treponema denticola by method of polymerase chain reaction. Claimed set includes specific to fragment of gene licCA of microorganism Treponema denticola primers 5'-TAG CCG GAA AAA CGA AGG AGT G-3' and 5'-CCC TGC TTG TTT GCA AAC ATA G-3', as well as probe (BHQ1)-5'-AAC CCA GCC G(FdT)T TCG TCC TCC GAC-3'-P, where BHQ1 stands for attached to 5'-tail nucleotide blank fluorescence damper, FdT - fluorescent dye FAM, attached to T nucleotide.

EFFECT: claimed invention represents an effective marker for identification of microorganism Treponema denticola presence in biological material.

FIELD: chemistry.

SUBSTANCE: invention relates to field of biochemistry, in particular to set of synthetic oligonucleotides for identification of DNA of parodontopathogenic microorganism Candida albicans by method of polymerase chain reaction. Claimed set includes specific to fragment of gene gr1 of microorganism Candida albicans primers 5′-TTGCCATTCTTGGACGAAGG-3′ and 5′-CAACAATGGCAACTTTTTTAGG-3′, as well as probe (BHQ1)-5′-TCCTCCTTCAG(FdT)CCCTGGTGCTGA-3′-P, where BHQ1 stands for attached to 5'-tail nucleotide blank fluorescence damper, FdT - fluorescent dye FAM, attached to T nucleotide.

EFFECT: claimed invention represents an effective marker for identification of microorganism Candida albicans presence in biological material.

FIELD: medicine.

SUBSTANCE: invention refers biotechnology and medicine. What is presented is a method based on measuring interleukin IL1B, IL8, IL10 and IL18 gene iRNA expression in vaginal smears in relation to the presence of iRNA of the reference genes B2M, GUS, TBP or HPRT; the derived expressions are used to calculate canonical linear discriminant function (CLDF) as follows: Y=1.09*IL1B-0.61*IL8+0.21*IL10-0.11*IL18-0.91 (formula 1), wherein IL1B is relative IL1B expression, IL8 is relative IL8 expression, IL10 is relative IL10 expression, IL18 is relative IL18 expression; IL=2^(Cpmin-Cpil)/NF (formula) wherein IL is relative interleukin gene expression, Cpmin is a coefficient of minimum expression, for IL1B Cpmin=17.9; IL8 Cpmin=16.6; IL10 Cpmin=28.8; IL18 Cpmin=23.3; Cpil is a threshold cycle of related IL in a sample determined automatically; NF is a rate setting factor calculated by formula 3: NF=4NFgus*NFhprt*NFb2m*NFtbp (formula 3) wherein NF is a rate setting factor calculated as a geometrical mean of 4 rate setting factors for reference genes (see formula 4); NFref=2^(Cpmin-Cpref) (formula 4), wherein NFref is a rate setting factor for the reference gene, Cpmin is a coefficient for minimum expression of the reference gene, Cpref is the threshold readings in the sample; Cpmin for the reference genes are as follows: GUS Cpmin=26.5; HPRT Cpmin=26.8; B2M Cpmin=18.9; TBP Cpmin=28.4; if CLDF≤0.1, the absence of vaginitis is stated; CLDF>0.1 stands for vaginistis.

EFFECT: invention enables the objective detection of the presence of vaginitis in a pregnant woman.

2 cl, 1 dwg, 3 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology. Claimed test-system for identification of RNA virus of bluetongue includes serogroupspecific primers and DNA-probe, complementary sites of conservative 10-th segment, which have the following nucleotide composition (5' - 3'): BTV/10/qf - ACKggTgCWACgCAAACACA; BTV/10/z - FAM - AARgCTgCATTCgCATCgTACGC - BHQ1; BTV/10/qr - ACRTCATCACgAAACgCTTC.

EFFECT: test-system possesses high sensitivity, specificity and quickness in carrying out analysis and makes it possible to identify RNA of bluetongue virus of serotypes by means of RT-PCR method in real time mode.

2 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, microbiology, and concerns the recovery and identification of pseudotuberculosis and intestinal yersiniosis agents (Y. Pseudotuberculosis and Y. Enterocolytica). A nutrient medium contains microbiological agar (dry), lactose, glucose, urea, calcium chloride, 1% alcoholic phenol red, 1% alcoholic methylene blue and distilled water in specific proportions.

EFFECT: invention enables reducing the length of studies.

3 dwg, 2 tbl

Up!