Recombinant strain of escherichia coli tg1(prvmoscow3253g-l) for obtaining set of pcr-standards and set of pcr-standards for determination of concentration of rabies virus "moskva 3253" in rabies antigen

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

 

The invention relates to biotechnology and for the construction of recombinant Escherichia coli strain carrying the cloned sequence fragment G-L region of the genome of strain of rabies virus "Moscow 3253" and set a panorama of standards for quantitative PCR, and can be used in the production of rabies immunoglobulin.

Unfavorable epidemiological situation of rabies, existing in almost all the regions of the Russian Federation due to the increase in the incidence of wild and domestic animals causes a sudden increase in the numbers of people each year seeking rabies through that dictates the need to improve rabies drugs, improving their quality [4].

Rabies immunoglobulin is obtained from hyperimmune serum producers immunized rabicheskogo antigen. The lack of quantitative methods for the determination of rabies virus in rabicheskogo the antigen for immunization prevents standardization of antigenic material.

One of the promising approaches to the definition of fixed rabies virus strain Moscow 3253" material for immunization is the use of quantitative polymerase chain reaction with hybridization-fluorescence analysis in real-time, the application of PCR-standards with a certain number of genome equivalents of viral particles or fragments of the genome of the virus.

Known PCR standards (EBVQ-PCR Standard, cat. No. STD020; HSV1Q-PCR Standard, cat. No. STD031; HSV2Q-PCR Standard, cat. No. STD032; HHV6Q-PCR Standard, cat. No. STD036), representing a solution of plasmids with different concentrations to determine the amount of virus Epstein-Barr, simple herpes virus first and second type, other viruses in biological material by PCR based on the results in real time, which are produced by NaNogen Advanced Diagnostics (Italy) [10, 12].

Known PCR standards (Colibrator) for the quantitative diagnosis of hepatitis C (cat. No. 01N30-070), HIV-1 (cat. No. 6L18-70), hepatitis b (cat. No. 02N40-70) and other PCR based on the results in real-time, produced by Abbott Molecular (USA) [2, 3]. However, PCR standards for the quantitative determination of rabies virus by PCR based on the results in real time, these firms are not released.

According to the results of searching for information on the availability of PCR standards for quantitative accounting of fixed rabies virus strain Moscow 3253" material for immunization by PCR based on the results in real-time is not found.

To create PCR standards used in the formulation of quantitative PCR, based on the results in real time producing strains of specific fragment specific region of the genome of rabies virus. N the date, there is no information about recombinant strains with a specific fragment region of the genome of rabies virus to quantify them.

To construct a recombinant strain required choice the unique region of DNA targets with high stability in all the passages of the virus required in production conditions and sufficient specificity.

The literature describes the use of target DNA N-region of the viral genome. This locus is characterized by a high conservatism [1]. There have been several alleles of this locus, which are specific to certain genotypes lyssaviruses[6, 7, 8, 9, 11, 13]. When studying the variability of the N-locus of rabies virus shows that this site may not be used as a matrix to determine using amplication technologies separate strains of the virus. To identify by PCR with reverse transcription cDNA fixed rabies virus strain Moscow 3253" as the DNA target best fits the G-locus and G-L region of the genome of rabies virus, characterized by high polymorphism and the incidence of mutations [1], which differs in certain strains of rabies virus.

According to the results of the patent search and literature data, information about the presence of recombinant strains carrying a fragment of the G-L region of the genome of rabies virus strain Moscow 3253"not found.

The present invention is to construct the strain is E. coli carrying the recombinant plasmid with the cloned sequence fragment G-L region of the genome of fixed rabies virus strain Moscow 3253", to obtain PCR standards and creation on its basis of a set of PCR standards for the quantitative determination of cDNA rabies virus strain Moscow 3253" in rabicheskogo the antigen used for production of rabies immunoglobulin, PCR based on the results in real time.

The technical result is to standardize stage of the technological process of making rabicheskogo antigen in the production of heterologous rabies immunoglobulin, by creating a recombinant strain of E. coli KM 229 for the production of PCR standards and set PCR standards based on it, allowing quantify rabicheskogo antigen by PCR with hybridization-fluorescence detection in real-time.

The technical result is achieved by the fact that the constructed recombinant E. coli strain carrying plasmid pRVMoscow3253G-L, containing a fragment of the G-L region of the genome of fixed rabies virus strain Moscow 3253", having a size 881 BP, the sequence SEQ ID NO1, designed to obtain a set of PCR standards.

The sequence SEQ ID NO1 presented in figure 1.

To design the simulation recombinant strain selected nucleotide sequence fragment G-L region of the genome of strain Moscow 3253" size 881 n / a, flanked by the primers BeshG 5'-gacttgggtctcccgaactgggg-3' and BeshL 5'-caaaggagagttgagattgtagtc-3' (5543 on 4663 nucleotide sequence of the genome of the virus strain RV-97, NCBI GenBank No. EF542830).

The cloning process consisted of the following steps.

1. Amplificatory fragment was purified from nevklyuchenie in the reaction of mononucleotides in the column "Gentry-sep colounins" (Pirseton Separations INC, USA) in accordance with the instructions of the manufacturer.

2. The fragment is ligated into the plasmid pGem-T using a commercial kit pGem-T Vector systems (Promega, USA) according to manufacturer's instructions.

3. Spent the transformation of cells of the strain E. coli TG1 by aliquot subsidized mixture. For this to 20±5 µl legirovannoi mixture was added 200±30 ál of freshly prepared competent cells. After incubation at 42°C for 90 seconds, the mixture was stirred on ice for 2 minutes and pokasivali 1 hour at 37°C. Cells were sown on agar medium with ampicillin at a concentration of 100 μg/ ml in Petri dishes and incubated for 18±2 hours at 37°C.

4. Grown colonies were checked for the presence of the fragment of the G-L region of the genome of fixed rabies virus strain Moscow 3253"having the sequence SEQ ID NO1, PCR with primers BeshG 5'-gacttgggtctcccgaactgggg-3' and BeshL 5'-caaaggagagttgagattgtagtc-3'5.

Colonies of E. coli TG1 (pRVMoscow3253G-L) were grown in liquid nutrient medium LB with ampicillin in test tubes for 18±2 hours at 37 ° C while on tandom the rocking on the rocking chair. The obtained biomass was collected by centrifugation in a micro test tubes in a volume of 1.5 ml

6. Provided the plasmids from the obtained biomass by the membrane filtration method using a set PureYield Plasmid Miniprep systems (Promega, USA) or equivalent.

7. Confirmed the presence of a fragment by PCR with primers BeshG and BeshL, specificity was evaluated by sekvenirovanie on a genetic analyzer, for example "SEO 800", using standard protocols for sample preparation and software of the device.

The recombinant strain carrying plasmid pRVMoscow3253G-L, containing a fragment of the G-L region of the genome of fixed rabies virus strain Moscow 3253" size 881 PN having the sequence SEQ ID NO1, characterized by the following features.

Cultural and morphological properties: gram-negative bacilli, which results in a nutrient medium LB with ampicillin smooth, with smooth edge of the colony diameter (2+0,5) mm

Physiological and biochemical properties: maintains the basic cultural-morphological and biochemical properties of E. coli TG1.

Plasmid pRVMoscow3253G-L with a cloned fragment of the G-L region of the genome of fixed rabies virus strain Moscow 3253"having the sequence SEQ ID NO1, is the starting product for obtaining PCR-standards used when the number is the result of PCR analysis in real-time to determine the number of fixed rabies virus strain Moscow 3253" in rabicheskogo the antigen in the production of rabies immunoglobulin.

The claimed recombinant E. coli strain TG1 (pRVMoscow3253G-L) deposited in the Public collections of pathogenic bacteria at FCUS antiplague research Institute "Microbe", number 229 KM.

The obtained recombinant strain of Escherichia coli KM 229 is used for obtaining a set of PCR standards used in the quantitative determination in PCR based on the results in real-time fixed rabies virus strain Moscow 3253" in rabicheskogo the antigen in the production of rabies immunoglobulin.

The technical result is also achieved by obtaining on the basis of plasmids pRVMoscow3253G-L, produced by recombinant E. coli strain KM 229, set PCR standards. PCR standards represent the solution of DNA plasmids pRVMoscow3253G-L in concentration from 108up to 103GE/ml

The set is for the quantitative determination of cDNA fixed rabies virus strain Moscow 3253" in rabicheskogo the antigen consists of PCR standards abbreviation (Rabies virus) RV1, RV2, RV3, RV4, denoting the concentration of DNA plasmids: RV1 - maximum concentrations up to RV4 - minimal. For the detection of high concentrations of rabies virus using a combination of PCR standards RV1, RV2, RV3; for determination of low concentrations - a combination of PCR standards RV2, RV3, RV4.

PCR standards RV1, RV2, RV3, RV4 was prepared from DNA solution pRVMoscow3253G-L, con is entrely which was determined spectrophotometrically at wavelengths of 260 and 280 nm. The optical density D=1 corresponds to 50 ág/ml double-stranded DNA. The purity of the DNA preparation was determined by the ratio of optical density of the solution at 260 and 280 nm. For preparation of PCR standards used the original DNA solution plasmids pRVMoscow3253G-L with a concentration of not less than 10 μg/ml at a ratio of D260/D280not less than 1.5.

Quantitative characterization of PCR standards RV1, RV2, RV3, RV4 expressed as the number of genome equivalents plasmids pRVMoscow3253G-L in the solution, which was calculated by the formula:

KpRVMoscow3253G-L=A×0,234×1012[GE/ML],

Where a is the concentration of DNA plasmide pRVMoscow3253G-L ág/ml.

PCR standard RV1 contains n×108GE/ml pRVMoscow3253G-L

PCR standard RV2 contains n×107GE/ml pRVMoscow3253G-L

PCR standard RV3 contains n×105GE/ml pRVMoscow3253G-L

PCR standard RV4 contains n×103GE/ml pRVMoscow3253G-L

The kit is intended for the quantitative determination of cDNA fixed rabies virus strain Moscow 3253" in vaccinated material on the stages of cooking rabicheskogo antigen used in the production of heterologous rabies immunoglobulin.

The set allows you to control and standardize manufacturing operations carried out at the stage of preparation rabicheskogo antigen in the production of g is teologiczne rabies immunoglobulin, that meets the requirements of GMP to conduct process and contributes to the improvement of the quality of the drug.

The practical application of PCR standards established on the basis of recombinant strain is illustrated by the following examples.

Example 1. Quantitative determination of high concentrations of rabies virus strain Moscow 3253" using a set of PCR standards for example rabicheskogo antigen organ or tissue of origin.

Rabicheskogo antigen organ or tissue of origin was obtained from brain suspension rabbits, which have previously entered the rabies virus strain Moscow 3253". Of antigenic material (the brain suspension rabbits), decontaminated according MU 1.3.2569-09 [5], prepared the samples for analysis.

Determining the concentration of rabies virus was performed in several stages.

Sample rabicheskogo antigen organ or tissue of origin was collected in 100 μl and transferred in 5 microcentrifuge tubes with a volume of 1.5 ml in Addition to a separate microcentrifuge tube 1.5 ml made of 100 ál of TE-buffer negative control DNA/RNA (MCI).

The allocation of RNA. For RNA extraction used "set of reagents for isolation of RNA/DNA from clinical material "RIBO-Sorb"manufactured by company (LLC Interlabservice Russia). Set reagent is for RNA extraction consists of lytic solution, solution for wash 1 solution wash 3, solution for cleaning 4, sorbent and RNA-buffer. 5 microcentrifuge tubes with 100 µl decontaminated rabicheskogo antigen organ or tissue of origin, and 1 tube MCI) was added to 450 µl of pre-heated at a temperature of (62±2)°C for 5-10 min lyse solution and thoroughly mixed. Was left to stand for 5-8 minutes, stirring 3-4 times. Centrifuged for 5 s at 3000-5000 rpm Sorbent carefully resuspendable and was added to 30 μl in each tube. Was stirred, left to stand for 1 minute, once again mixed and left for 5 min Centrifuged tubes at 10,000 rpm for 45 C. the Supernatant was removed, and to the precipitate was added to 400 μl of the solution for washing 1. Was stirred until complete resuspendable sorbent, centrifuged for 45 s at 10000 Rev/min After removal of supernatant was added to the tube with 500 µl of solution for cleaning 3. Sorbent carefully resuspendable and the tubes were centrifuged for 45 s at 10000 Rev/min After removal of the supernatant operation is repeated again. Then in a test tube was added 400 μl of solution for cleaning 4, carefully resuspendable and centrifuged for 45 s at 10000 rpm the Supernatant was removed and tubes were placed in a thermostat at 60°C for 10 minutes, with the lid on toulali open.

After drying in each tube was added 50 μl of RNA-buffer, was mixed and placed in a solid-state thermostat at 60°C for 3 min was Mixed and centrifuged for 1 min at 13,000 rpm the Supernatant contained the purified RNA virus.

Carrying out the reaction of reverse transcription.

For the given reaction reverse transcription used the kit reagents to obtain cDNA on the RNA matrix "Reverta-L" (Interlabservice LLC", Russia). The kit reagents to obtain cDNA on the RNA matrix contains RT-G-mix-1, RT-mix - 0,125 ml, revertase (MMLv) and DNA-buffer.

Before you begin from the freezer extracted tubes RT-mix and RT-G-mix-1 (RT-G-mix-2) and thaw the contents of the test tubes at room temperature. They are then thoroughly mixed, and centrifuged for 5 s at 3000-5000 rpm for deposition of droplets from the walls. To prepare the reaction mixture in a test tube with RT-mix was made in 5 ál of RT-G-mix-1 (RT-G-mix-2), thoroughly mixed, centrifuged for 5 s at 3000-5000 rpm for deposition of droplets from the walls. To the resulting solution was added 6 μl of revertase MMLv, gently mixed and centrifuged for 5 s at 3000-5000 rpm for deposition of droplets from the walls.

Took 5 microcentrifuge tubes with a volume of 0.6 ml, corresponding to the number of samples tested and RCC. In each tube to make the and 10 μl of the resulting reaction mixture, then in a test tube was added 10 μl of a preparation of RNA and 10 μl of TE buffer in MCI. The tubes were incubated in the amplifier or solid state thermostat for 30 min at 37°C. all tubes were added to 20 ál of DNA-buffer and gently 8-10 times mixed. The obtained cDNA was used for PCR with hybridization-fluorescence in light of the results.

Staging PCR.

From the freezer extracted microtube with a volume of 0.2 ml containing PCR-mix - 1, PCR-mix - 2, TE-buffer, the enzyme Taq DNA polymerase with an activity of 5 u/µl, a set of PCR standards RV1, RV2, RV3 to account for high concentrations of antigen, rabies virus in the sample. The contents of the tubes were completely thawed.

PCR-mix-1 in a volume of 5 µl contained a solution of primers RV8 5'-ACGTACTATGACCTCTACCC-3', RV9 5' - CCAGGAACTAATACTAAGGG-3' and probe RV10 5'-FAM-CTCCGATGATCCCATGCTTG-BHQ1-3', providing amplification of the fragment of the G-L region of the genome of fixed rabies virus strain Moscow 3253", 8 and 4 pmol, respectively, 1 mmol of each of the four dNTP, 0.025% sodium azide and water, free from nucleases. PCR-mix-1 transferred 5 ál 9 microcentrifuge tubes with a volume of 0.2 ml In each tube on the surface of the PCR-mix-1 was layered molten 20% solution of paraffin wax in mineral oil.

PCR-mix-2 in a volume of 10 µl, containing 2.5×Taq buffer with ammonium sulfate, and 6.25 mmol m is fester chloride, the enzyme Taq DNA polymerase, water, free from nucleases, was stirred at microcentrifuge/shaker and transferred to 10 μl of a 5 micro test tubes with PCR-mix-1 without damaging the layer of wax.

5 micro test tubes were made in 10 μl of sample cDNA, 3 microtube was made on 10 µl of each PCR standard RV1, RV2, RV3, 1 microprobing contributed 10 ál of MCI-samples containing THE buffer, the study of which is analyzed together with the samples from step DNA extraction. 1 microprobing with a negative control of amplification ( -) contributed 10 μl of TE buffer.

Prepared microtube was placed in thermal cycler RotorGene 6000 and performed PCR with hybridization-fluorescence in light of the results when the mode annealing of primers at a temperature of 55°C for 10 cycles, then at 51°C for 35 cycles. Accounting fluorescence channel FAM carried out at a temperature of 51°C.

Accounting results.

Quantitative assessment of the content of cDNA of rabies virus in the antigenic material was carried out according to the value of the fluorescence intensity signal by comparing the sample with PCR standards RV1, RV2, RV3 using software thermal cycler RotorGene 6000, in the presence of a negative result in the samples and MCI.

The concentration of fixed rabies virus strain Moscow 3253" in 5 samples of brain suspension kr the faces ranged from 5,57×107 copies/ml to of 3.07×109 copies/ml (figure 2). Values of the correlation coefficient was 0,99086, the reaction efficiency is 0,85949.

Example 2. Quantitative determination of low concentrations of rabies virus strain Moscow 3253" for example, the culture fluid at the stage of growing the virus in cell culture using a set of PCR standards.

Were selected for the study 6 samples vaccinated culture liquid obtained in the suspension method of growing Virus fixe on the culture of Vero cells to the stage of concentration. Disinfection vaccinated material, isolation of RNA, reverse transcription reaction and PCR was carried out similarly to example 1, except when conducting PCR used a set of PCR standards RV2, RV3, RV4 to account for low levels of rabies virus in the sample.

The concentration of fixed rabies virus strain Moscow 3253" in the analyzed samples ranged from 3,31×104 copies/ml up to 1.59×106 copies/ml (figure 3). Values of the correlation coefficient was 0,99959, the reaction efficiency is 0,90885.

Thus, the claimed recombinant E. coli strain TGl(pRVMoscow3253G-L) to obtain a set of PCR standards and a set of PCR standards for the quantitative determination of cDNA fixed rabies virus strain Moscow 3253" in rabicheskogo the antigen allow you to define different degrees of concentration of fixed rabies virus "Moscow 3253" in VI is ostergade material, to control and standardize manufacturing operations carried out at the stage of preparation rabicheskogo antigen in the production of heterologous rabies immunoglobulin, which meets the GMP requirements to conduct process and contributes to the improvement of the quality of the drug.

Literature

1. Gruzdev PHD, Nedosekov CENTURIES Rabies in animals. M.: 2001. 25-26 C.; 44 C.

2. Directory http://www.nadspa.com/ accessed on 23.05.2012,

3. Directory http://www.abbottmolecular.com/home.html date of access 23.05.2012,

4. Onishchenko GG ON sanitary and epidemiological situation in the Russian Federation in 2009: State report. M: Federal center of hygiene and epidemiology. 2010. 334-336 C.

5. Organization of work of the laboratories using methods of nucleic acid amplification when working with material containing microorganisms of I-IV groups of pathogenicity // HOWTO MU 1.3.2569-09, Moscow, 2009. - P.37-40.

6. Bordignon J., Brazil-dos-Anjos G., Bumo C.R. et al. Detection and characterization of rabies virus in Southern Brazil by PCR amplification and sequencing of the nucleoprotein gene // Arch Virol. - 2005. - Vol.150. - P.695-708.

7. Bourhy h, Kissi C., Tordo N. Molecular diversity of the Lyssavirus genus // Virology. - 1993. - Vol.l94. - P.70-81.

8. Dantasjunior J.V., Kimura L.M.S., Ferreira M.S.R. et al. Reverse reduced polymerase chain reaction assay for rabies virus detection // Arq. Bras. Med. Vet. Zootec. - 2004. - Vol.56. - P.398-400.

9. Heaton P.R., Johnsbone P., McElhinney L.M. et al. Heminested PCR assay for detection of six genotypes of rabies ad rabies-related viruses // J. Clin. Environ. - 1997. - Vol.35. - P.2762-2766.

10. Kosinova E. et al. Real-time PCR for quantitation of bovine viral diarrhea virus RNA using SYBR Green I fluorimetry. Veterinarian medicine, 52, 2007; 6: 259-261 p.

11. Nagaraj So, Vasanth J.P., A. Desai et al. Ante mortem diagnosis of human rabies using saliva samples: comparison of real-time and conventional RT-PCR techniques // J. Clin. Virol. - 2006. - Vol.36. - P.17-23.

12. Radonic, A., Thuike S. et al. Reference gene selection for quantitative real-time PCR analysis in virus infected cells: SARS corona virus. Yellow fever virus. Human Herpesvirus-6, Cameplox virus and Cytomegalovirus infections. Virology. 2005; 7: Tab2.

13. Saengseesom W., Mitmoonpitak C., Kasempimolpom s, Sitprija V. Real-time PCR analysis of dog cerebrospinal fluid and saliva samples for ante-mortem diagnosis of rabies // Southeast Asian J. Trop. Med. Public Health. - 2007. - Vol.38. - P.53-57.

1. Recombinant strain ofE. coliTG1(pRVMoscow3253G-L) to obtain a PCR standards for the quantitative determination of cDNA strain Moscow 3253", based on the strain ofE. coliTG1 by transformation with plasmid pRVMoscow3253G-L, obtained by legirovaniem fragment of the G-L region of the genome of fixed rabies virus strain Moscow 3253"having the sequence SEQ ID NO1, in plasmid pGem-T deposited in the Public collections of pathogenic bacteria "Microbe", number 229 KM.

2. A set of PCR standards for the quantitative determination of different concentrations of fixed rabies virus strain Moscow 3253" in vaccinated material by polymerase chain reaction with hybridization-fluorescence in light of the results, containing solutions of DNA plasmids pRVMocow3253 G-L, bearing a fragment of the G-L region of the genome of fixed rabies virus strain Moscow 3253"having the sequence SEQ ID NO1, at concentrations of 108, 107, 105, 103GE/ml



 

Same patents:

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: biotechnology.

SUBSTANCE: invention is production of the nutrient medium, which creates optimal conditions for growing legionella, comprising: enzymatic hydrolyzate of pig lung, enzymatic hydrolyzate of chicken egg yolk, potassium monophosphate, trihydrate disubstituted potassium phosphate, L-cysteine hydrochloride, activated carbon, microbiological agar and distilled water at a predetermined ratio of ingredients.

EFFECT: invention enables to produce the high-quality, easy-to-prepare nutrient medium, to reduce the time of growing legionella.

FIELD: biotechnologies.

SUBSTANCE: nutritive medium includes lactoserum, yeast autolysate, acetic acid 70%, agar and cabbage brew at the specified component ratio.

EFFECT: invention allows increasing selectivity of nutritive medium and simplifying its production.

8 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used in biological treatment of waste water from electroplating plants from heavy metal salts. The method involves adding yeast biomass to waste water, said biomass being in form of brewery wastes containing a combination of yeasts of different strains of Saccharomyces cerevisiae with viability of 90-95% in a given amount. The biomass is mixed with the waste water to obtain a suspension. The obtained suspension is held for 8 hours at temperature of 10°C-29°C and solution pH of 5.5-8.0, followed by recycling spent yeast containing heavy metals by treating with lime Ca(OH)2, with the ratio of yeast biomass to lime of 1:5-8, to obtain a mixture. The obtained mixture is subjected to wet treatment at temperature of 90°C for 1 hour, followed by isolation of the obtained mixture, which contains heavy metals, in concrete paste.

EFFECT: invention increases efficiency of purifying waste water from heavy metal ions.

3 tbl, 3 ex

FIELD: biotechnologies.

SUBSTANCE: invention presents a diagnostics method of sensitivity of M. tuberculosis (MBT) to injection antituberculous preparations of a reserve row. The method involves a DNA extraction stage, amplification of the investigated DNA sections by means of a polymerase chain reaction method and analysis of conformational polymorphism of single-chain fragments (SSCP). Gene section rrs is amplified in 50 mcl of a reaction mixture with addition of 5 mcl of the specimen. Promoter gene section eis is amplified in 30 mcl of the reaction mixture containing direct 5'CGGAGCCGTCGGGGTATGC and reverse 5'GCCGCGGCCAGTAGGAACA primers and 3 mcl of the specimen as per the amplification programme: 1-st stage - 95°- 4 min; 2-nd stage - 95° - 20 sec, 59° - 30 sec, 72° - 20 sec (30 cycles); 3-rd stage: 72° - 4 min; 10° - storage. Separation of amplification products in the ratio of 4 mcl of the specimen and 6 mcl of a denaturing dye is performed by electrophoresis in 8% polyacrylamide gel with 5% glycerin at voltage of 400 Volts during 5 hours at 8°C. Gel painting is performed by means of caustic silver.

EFFECT: invention allows diagnosing sensitivity of M tuberculosis to injection antituberculous preparations of a reserve row with high accuracy.

2 dwg

FIELD: biotechnologies.

SUBSTANCE: invention proposes an association of strains of bacteria-oil decomposers, which have been extracted from oil-contaminated soil, Acinetobacter species B-1037, Pseudomonas species B-989, Bacillus species B-1040, deposited at The State Research Centre of Virology and Biotechnology VECTOR. Besides, at least 30% bacteria of each strain is contained in the association. Remediation of oil-contaminated soils includes water suspension of lyophilic dried biomass of the strain association based on 109 cells per square metre. Strains of the association can utilise a wide range of oil components at the temperature of 10-15°C.

EFFECT: improving cleaning efficiency of oil-contaminated soils.

2 cl, 5 dwg, 2 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: bacillus subtilis subsp.subtilis BKM B-2711D strain has apparent antagonism in relation to Escherichia coli, Salmonella typhi, Staphylococcus aureus, Listeria monocytogenes, and resistance to streptomycin and tetracycline antibiotics. It is deposited in the All-Russia Collection of Microorganisms of the Institute of Biochemistry and Physiology of Microorganisms Named after G.K. Skryabin of the Russian Academy of Science (IBFM RAN) and has the following registration number: BKM B-2711D, and can be used at production of probiotic bacterial preparations that can be used in veterinary medicine.

EFFECT: invention allows increasing protease and xylanase activities.

1 tbl, 3 ex

FIELD: biotechnologies.

SUBSTANCE: bacillus amyloliquefaciens BKM B-2714D strain has apparent antagonism in relation to Salmonella typhi, Staphylococcus aureus, Listeria monocytogenes, and resistance to tetracycline and trimethoprim antibiotics. It is deposited in the All-Russia Collection of Microorganisms of the Institute of Biochemistry and Physiology of Microorganisms Named after G.K. Skryabin of the Russian Academy of Science (IBFM RAN) and has the following registration number: BKM B-2714D, and can be used at production of probiotic bacterial preparations that can be used in veterinary medicine.

EFFECT: invention allows increasing protease and xylanase activities.

1 tbl, 3 ex

FIELD: biotechnologies.

SUBSTANCE: invention can be used for detection of coliform bacteria and E.coli in specimens of food products and water at performance of bacteriological tests. Feed medium includes a nitrogen source represented by meat peptone or pancreatic hydrolysate of fish flour, sodium chloride, dibasic sodium phosphate, potassium monophosphate, sodium pyruvate, L-tryptophane, sodium dodecyl sulphate, 6-chloro-3-indolyl-β-D-galactopyranoside (Salmon - GAL), 5-bromine-4-chloro-3-indolyl-β-D-glucoronide-(X-GLUC), isopropyl- β-D1-tiogalactopyranoside (IPTG) and microbiological agar in the specified ratio.

EFFECT: invention allows reducing identification time, improving difernetiation accuracy of coliform bacteria and Ecoli, and simplifying a feed medium preparation method.

2 cl, 4 dwg, 1 tbl, 3 ex

FIELD: biotechnologies.

SUBSTANCE: invention can be used for detection and considering of E.coli and coliform bacteria in water, food products, clinic material, etc. Feed medium contains pancreatic hydrolysate of fish flour dried with Tergitol 7 on the bases of 0.1 g of Tergitol 7 per 6 g of dry pancreatic hydrolysate of fish flour, yeast extract, 1-water D (+) lactose, bromthymol blue, sodium dodecyl sulphate, 2,3,5-triphenyltetrazolium chloride, sodium carbonate and microbiological agar in the specified ratio.

EFFECT: invention allows preserving sterility of feed medium during 10 days, increasing accuracy of differentiation of coliform bacteria and Ecoli and simplifying a feed medium preparation method.

2 tbl, 4 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: chemistry.

SUBSTANCE: invention relates to biotechnology and specifically methods of forecasting development of cancer and can be used in medicine. The method of performing forecast for a patient suffering from NSCLC involves determining the expression level of ChoK beta or ChoK beta and ChoK alpha in a sample collected from said patient. Low levels of ChoK beta relative to the levels in a standard sample indicate a bad forecast for the patient. Low levels of ChoK alpha and high levels of ChoK beta relative to the expression levels of said proteins in the standard sample indicate a good forecast for the patient.

EFFECT: invention enables to forecast probability of survival, for example, survival without relapses for a patient with NSCLC.

6 cl, 16 dwg, 2 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to oncology and molecular biology. What is presented is a method for determining the non-small cells lung cancer sensitivity to the preparations reactivating protein p53, involving the recovery of RNA from samples, the synthesis of complementary DNA of the genes CDKN1A, BTG2 and E2F1 by reverse transcription and real-time polymerase chain reaction, and the determination of a relation of the amount of complementary DNA of the gene E2F1 to the amount of complementary DNA of the gene CDKN1A or the gene BTG2, wherein if observing the relation of E2F1/CDKN1A>3 or E2F1/BTG2>1.5, the non-small cells lung cancer cells are considered to be sensitive to the preparations reactivating protein p53.

EFFECT: invention may be used in treating oncological diseases.

3 dwg, 3 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to molecular biology and can be used in diagnostic studies aimed at detecting acute intestinal infection (AII) agents. Disclosed is a set of differentiating oligonucleotides (probes), which enables to determine, in a biological sample, DNA of pathogenic microorganisms which cause AII, and specific identification thereof, where said microorganisms relate to a group which includes Shigella spp. and Enteroinvasive E.coli (EIEC), Salmonella spp., Campylobacter Jejuni, Proteus mirabilis, Klebsiella pneumoniae.

EFFECT: described is design of a biochip on which is immobilised a set of probes disclosed herein, designed to perform rapid analysis of AII agents in clinical samples and material obtained from environmental media.

2 cl, 4 tbl, 2 dwg, 5 ex

FIELD: biotechnologies.

SUBSTANCE: invention presents a diagnostics method of sensitivity of M. tuberculosis (MBT) to injection antituberculous preparations of a reserve row. The method involves a DNA extraction stage, amplification of the investigated DNA sections by means of a polymerase chain reaction method and analysis of conformational polymorphism of single-chain fragments (SSCP). Gene section rrs is amplified in 50 mcl of a reaction mixture with addition of 5 mcl of the specimen. Promoter gene section eis is amplified in 30 mcl of the reaction mixture containing direct 5'CGGAGCCGTCGGGGTATGC and reverse 5'GCCGCGGCCAGTAGGAACA primers and 3 mcl of the specimen as per the amplification programme: 1-st stage - 95°- 4 min; 2-nd stage - 95° - 20 sec, 59° - 30 sec, 72° - 20 sec (30 cycles); 3-rd stage: 72° - 4 min; 10° - storage. Separation of amplification products in the ratio of 4 mcl of the specimen and 6 mcl of a denaturing dye is performed by electrophoresis in 8% polyacrylamide gel with 5% glycerin at voltage of 400 Volts during 5 hours at 8°C. Gel painting is performed by means of caustic silver.

EFFECT: invention allows diagnosing sensitivity of M tuberculosis to injection antituberculous preparations of a reserve row with high accuracy.

2 dwg

FIELD: biotechnologies.

SUBSTANCE: as per the first version, a method is implemented by recording of cyclic voltamperograms of a working electrode modified with carbon nanotubes with a oligonucleotide probe noncovalently immobilised on their surface, before and after the nucleic acid specimen is added to the investigated solution, and as per the change of capacitive characteristic, it is evaluated whether a section complementary to the oligonucleotide probe is available in the specimen or not. The second version of the method differs by the fact that non-covalent immobilisation of the oligonucleotide probe onto the surface of nanotubes is performed by means of an anchor group pre-introduced to the probe. This version includes recording not only of the change of surface area of voltamperograms from cycle to cycle, but also occurrence of a specific peak on a cyclic voltamperogram, which is related to fixation of detected nucleic acid complete with the modified probe. Intensity of the peak on the cyclic voltamperogram is pro rata to concentration of the determined nucleic acid, which allows performing quantitative evaluation. A device for implementation of the detection method of specific sequences of nucleic acids represents an electrochemical analyser that consists of a three-electrode electrochemical cell, the electrodes of which are connected to a recording device, and the working electrode is made from a silicone substrate modified with vertically oriented carbon nanotubes with an immobilised oligonucleotide probe complementary to the nucleic acid to be determined.

EFFECT: improving evaluation accuracy.

8 cl, 5 dwg, 4 ex

FIELD: biotechnologies.

SUBSTANCE: differentiation of four Trichobilharzia species: T. szidati, T.regenti, T.franki and T.sp.var.narochanica is performed by amplification of sections of sequence of nuclear ribosomal DNA (rDNA) in specimens of reproductive helmints, their larval stage and/or on rDNA of fresh-water mollusks of Lymnaeidae family, which are infected with the above helmints by PCR and four oligonucleotide primers of the following composition: F: 5'-CTTTCCATCTATCACGATGCACT-3' R1: 5'-ATGATAATGTGCATAACACACC-3' R2: 5'-GCCGTTTATTTATATGTATGTG-3' R3: 5'-CAAGCCGTTTATTWATATATAACGG-3'. The obtained amplification products are visualised and differentiated and identified as per size (length); with that, one amplification fragment with the size of 255 base pairs is relevant for T.regenti, species, one fragment with the length of 316 base pairs is relevant for T.szidati species, two fragments with the size of 255 and 316 base pairs are relevant for T.sp.var.narochanica species, and amplification fragment with the size of 258 base pairs is detected only for T.franki species.

EFFECT: method allows simultaneous detection and species identification of fours types of ornithic schistosomes of Trichobilharzia species at different life stages.

2 dwg, 2 tbl, 2 ex

FIELD: veterinary science, virology, biotechnology.

SUBSTANCE: the suggested canine rabies vaccine contains a plasmid that contains a nucleic acid that codes rabies virus protein G. The vaccine provides complete protection against rabies for the period of 1 yr after a single injection of the vaccine suggested. It has been, also, suggested the method for vaccination with the help of such a vaccine and a kit that includes this vaccine.

EFFECT: higher efficiency of vaccination.

24 cl, 10 dwg, 19 ex

Up!