Test-system for identification of rna virus of bluetongue by rt-pcr method in real time mode |
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IPC classes for russian patent Test-system for identification of rna virus of bluetongue by rt-pcr method in real time mode (RU 2510851):
 Methods of treating and diagnosing cancer / 2509809
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.
 Method for determining non-small cells lung cancer sensitivity to preparations reactivating protein p53 / 2509808
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.
 Set of differentiating and specific oligonucleotides for identifying dna of acute intestinal infection agents, method of identifying acute intestinal infection, microchip and diagnostic system for carrying out method / 2509804
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.
 Diagnostics method of sensitivity of mycobacterium tuberculosis strains to injection antituberculous preparations of reserve row (aminoglycosides and capreomycin) / 2509158
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.
 Detection method of specific sequences of nucleic acids (versions), and device for its implementation / 2509157
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.
 Method of species dna differentiation at different stages of life cycle of helmints-excitants of cercarial dermatitis of human being / 2509156
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.
 Analysis method of eml4-alk translocations associated with sensitivity of lung cancer to antitumour target therapy / 2509153
Proposed method involves the following: obtaining EML4-ALK cDNA by means of a polymerase chain reaction with reverse transcription (RT-PCR) on RNA matrix of EML4-ALK gene using specific primers; amplification of fragments of EML4-ALK gene by means of a multiplex PCR method on cDNA matrix obtained at the first stage of RT-PCR by means of high-specific primers; obtaining fluorescent-marked PCR-product at the second stage of RT-PCR; creation of a biochip for analysis of EML4-ALK translocations, which contains a set of immobilised probes; hybridisation of fluorescent-marked PCR-product with probes in gel cells on a plastic substrate of the biochip; recording and interpretation of hybridisation results. The method provides for use of a technology of DNA biochips designed for the purpose of determining 6 versions of EML4-ALK translocations (V2, V3, V5, V4, V7, V1).
Method of quantitative evaluation of terminal nucleotides of g-chain of telomeric human rna by means of polymerase chain reaction and duplex-specific analysis, sets of synthetic oligonucleotide primers and probes for implementation of above said method / 2508407
Invention proposes a method involving stages of preliminary extraction of genomic RNA, extraction of a specific fraction of single-stranded G-overhanges of telomeric RNA and further amplification of their negative chain with duplex-specific analysis; besides, amplification stages include modification of 3'-ends of telomeric overhangs by means of terminal deoxynucleotidyl transferase and are implemented using a set of primers SEQ ID NO: 1-5. The duplex-specific analysis is based on hybridisation of minus chains of overhangs with a set of specific fluorescent marked probes SEQ ID NO: 6-11, which correspond to six possible versions of nucleotide endings of G-chain of telomeric human RNA. A buildup degree of probes in presence of a ferment of duplex-specific nuclease serves as a criterion of presence of the corresponding version of the nucleotide ending and a measure of its quantitative content. Obtained profiles of terminal nucleotides of G-chain of telomeric RNA serve as proliferative markers pointing at an activation degree of cell fission and determining time duration of a cellular cycle.
 Method of species identification of l.casei/paracasei, l.fermentum, l.plantarum and l.rhamnosus / 2508406
Invention refers to a method of species identification of L.casei/paracasei, L.fermentum, L.plantarum, L.rhamnosus lactobacilli. The proposed method involves performance of a PNR reaction with species-specific primers; besides, primers are built, which are specific to the first gene of operon FIFO ATP of synthase (a gene of subunit a) and a gene of uracylphosphoribosyltransferase, which precedes it, for L.casei/paracasei and L.rhamnosus and a gene of uracyltransport protein for L.plantarum and L. fermentum.
 Edwards syndrome determination technology by sequenation method / 2507269
Method for determination of foetus Edwards syndrome, where from pregnant women plasma extracted is extracellular DNA, it is subjected to disulfide conversion succeeded by polymerase chain reaction and further mass parallel sequenation of differential methylated sites; as control of differential methylated sites used are sites with various levels of methylation of a mother and a foetus, data are analysed by determination of relation of reading quantities obtained by sequenation of differential methylated sites of DNA on purpose and control chromosomes, where in case of trisomyunder constant quantity of foetus DNA readings, charted on differential methylated sites of chromosomes there will be observed quantity of readings charted on purpose chromosome to quantity of readings on control chromosomes equal to 3/2, while as a norm this relation will be constant equal to 1.
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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
The invention relates to biotechnology, namely, genetic engineering, and can be used in veterinary Virology for gene diagnostics, namely to detect RNA virus bluetongue. The bluetongue virus (WB), causing disease in domestic and wild ruminants, is one of the species of the genusOrbivirus,familyReoviridae[1]. The viral genome consists of 10 segments of dsrnas Packed in dodecahedral nucleocapsid, consisting of seven structural proteins [2]. Ten linear dsrnas segments of the genome WB vary in size from 3944 BP to 822 BP (for example WB serotype 8) [3]. They are marked as segments 1-10, in accordance with the decrease in molecular weight. Genome WB encodes 10 proteins of the virus, each protein is one of the segments of dsrnas [4]. Seven proteins (VP1-VP7) are structural components of the virus particle, the other three (NS1,NS2, and NS3) are non-structural proteins that are synthesized during replication of the virus in infected cells. The smallest of the nonstructural proteins, NS3, coded 10 segment is the fourth most variable protein WB [3], the analysis of which allowed us to separate isolates of the virus for a few treasures [3], however, it is considered to be moderately conservative relative to other segments. For example, the NS3 protein of the virus ACHL has an extremely high level of variations that phospholinoleate sequence 10 segment 3 cluster - alpha, beta and gamma [5]. Outside Africa outbreaks ACHL was relatively small and therefore the absence of any geographical grouping of viruses in 10 segment reflects the restriction of the virus in the African continent. NS3 is involved in the process of releasing particles WB from the infected host cell [6]. It is now known about the existence of 26 genotypes of the virus, of whom 25 are antigenically match 1-24 and 26 serotypes of the virus. The data of molecular epidemiology indicate a high genetic heterogeneity of its population, which allows you to split virus types, topotype, quanitity. At the moment we know more than 300 tons of virus variants, genetically different from each other. There is a need for improved means and methods of laboratory diagnostics, diagnosis in asymptomatic course of the disease and the emergence of new genetic variants of the virus, namely the development seroprotection test system to detect RNAbluetongue virus RT-PCR in real time, allowing a short time to establish the presence of the genome of the pathogen in samples of biological material from infected animals. Difficulties in its development due to several factors: firstly, the structure of the genome, as it presents the 10th segment of the mi dsrnas and as a result is currently known fact frequent reasontly viruses as field options field and with the vaccine, not only within the same serotype, but also between serotypes. Known quantitative method RT-PCR real-time detection of vaccine and/or field strains WB. Since the detection of PCR products was based on fluorescence intensity, which greatly increased the performance of the test and reduces the risk of contamination. There were developed two universal test based on RT-PCR in real time, they allowed us to detect all serotypes WB [7, 8, 9]. All test systems were developed abroad and for the period when it was aware of only 24 serotypes of the virus. A significant drawback is their high cost and long delivery time in Russia, and validation using only the 24 serotypes, which can lead to false-negative results in the study material that contains a virus bluetongue new genetic variants, or decrease the specificity of the sets. The aim of the present invention is to provide seroprotection test systems for RNA detection of bluetongue virus in samples of biological material based on amplification of the 10th segment of the genome of bluetongue virus. The set C is l is achieved by the proposed test system for the detection of the genome of bluetongue virus by polymerase chain reaction in real time includes synthetic oligonucleotide primers and DNA probes, complementary to the conservative areas of the 10th segment, having the following nucleotide composition(5' - 3'): BTV/10/qf - ACKggTgCWACgCAAACACA BTV/10/z - FAM - AARgCTgCATTCgCATCgTACGC - BHQ1 BTV/10/qr - ACRTCATCACgAAACgCTTC The technical result consists in increasing the efficiency of RNA detection of bluetongue virus in samples of different types of biological material, the ability to determine the serotype of bluetongue virus by PCR with the stage of reverse transcription in real time, as well as increased specificity and sensitivity, the reduction of time in determining the serotype of bluetongue virus in the samples of the studied biological material. The invention consists in that by means of these serogrouping oligonucleotide primers and DNA probe carried out RT-PCR in real time, allowing to detect RNA virus bluetongue 25 serotypes (1-24, 26). This system allows to increase the specificity and sensitivity of the reaction. In the basis of the method used RT - PCR real-time technology hybridization probes TaqMan controlling the kinetics of PCR directly in Ho is e amplification using resonance fluorescence quenching. For detection used a probe carrying a fluorophore and cositel, complementary part amplifierarava with specific primers fragment. Table 1 Seroprotection primers and DNA probes for the detection of RNA virus bluetongue
Selection and analysis serogrouping primers and probes (table. 1) was performed using the software package "Bio Edit http://www.mbio.ncsu.edu/bioedit/BioEdit_708_062707.zip", "Oligo 6.0" on the basis of the analysis of the nucleotide sequences of the 10th segment of the reference strains and isolates 26 (1-26) serotypes of the virus, published in GenBank. As a source of fluorescence on the 5' end of the probe used dye FAM, and t is the solution of fluorescence on the 3' end BHQ1. Specific example Detection of the genome of bluetongue virus in samples of biological material (blood from infected with bluetongue the animals, the cultural material of the virus, freeze-dried culture material of the virus from the Museum of strains of microorganisms wildebeest Vniivvim RAAS, as well as in samples of intact cell cultures, blood from intact animals and cultural material heterologous viruses). From the presented samples using reagent Trizol (Invitrogen) and according to manufacturer's instructions were isolated nucleic acid. Further preparations of nucleic acids used in the reaction: 8 μl of sample was added to the reaction mixture to denature. In a test tube with denatured RNA was added to the mixture for the reaction of reverse transcription. Drugs received complementary DNA in the amount of 10 μl was used for setting the actual PCR in real time (results are presented in table 2). Carrying out the reaction of reverse transcription In the prepared tubes contribute 7 ál of the mixture to denature, the top layer 40 μl of mineral oil. Under the oil make 8 ál of RNA preparation. The reaction denaturation is carried out at a temperature of 95°C for 5 minutes. Next, you need to freeze the mixture after denaturation, by placing the tubes in a rack with the chill is entom. Cook the mixture for reverse transcription for this one sample in a separate test tube is mixed with 9.5 μl of the mixture for reverse transcription and 0.2 μl (40 units) MMLV-revertase, the mixture is stirred. In tubes after denaturation contribute to 9.7 ál of the mixture for reverse transcription. Reverse transcription is performed at a temperature of 42°C for 30 minutes. After the reaction, the preparation of cDNA used for PCR. The PCR in real time In a separate tube, prepare the General reaction mixture for the required number of samples, one sample add 8 ál of the mixture for PCR, 7 μl of a mixture of primers and 0.2 μl (1 u) of Taq polymerase. The mixture is stirred, avoiding foam formation, and dig in 15 ál of tube a volume of 0.2 cm3on the surface of the mixture make the wax in the volume of 15 µl. In a report prepared for PCR tubes on wax make 10 ál of cDNA. The profile reaction: 1. Hold temperature 95°C - 3 min 2. The Cycling 95°C - 10 c 60°C - 20 72°S - 15 The cycle is repeated 5 times. 3. The Cycling with detection 2 95° C - 10 c 60° C - 20 C - Detection 72° S - 15 Cycle to repeat 40 times. The fluorescence measured in the Green channel at 60°C. Accounting and interpretation of results amplification The results are interpreted on the basis of the presence/absence of crossing the Riva fluorescence installed at the level of the threshold line (Threshold), which corresponds to the presence/absence values of Ctin the corresponding column in the results table. Table 2 Results detection of RNA virus bluetongue RT-PCR in real time using seroprotection test systems
Thus, the developed test system that allows using the method of RT-PCR in real time to detect RNA virus bluetongue 25 serotypes (1-24, 26). The main advantages of the claimed invention are: - high specificity and sensitivity of the test system for the detection of RNA virus bluetongue, thanks to selective detection of a unique nucleotide sequence of the 10-th segment of the viral genome; - the speed of analysis, as a number of procedures needed to register the amplification products in classical PCR; - easy-to-follow techniques, eliminating the need to attract highly qualified personnel; - relative to the positive cheapness of the method, through the use of RT-PCR in real time to identify the genome of the pathogen. Sources of information 1. The dsRNA viruses/ P.P.C. Mertens// Virus Res. - 2004. -Vol.101. - P.3-13. 2. Bluetongue virus assembly and morphogenesis/ P. Roy, R. Noad// Curr Top Environ Immunol. - 2006. - Vol.309. - P.87-116. 3. Sequence analysis of bluetongue virus serotype 8 from the Netherlands 2006 and comparison to other European strains/ S. Maan, N. S. Maan, N. Ross smith, C. A. Batten, A. E. Shaw, S. J. Anthony, A. R. Samuel, K. E. Darpel, E. Veronesi, C. A. L. Oura, K. P. Singh, K. Nomikou, A. C. Potgieter, H. Attoui, E. van Rooij, P. van Rijn, K. D. Clercq, F. Vandenbussche, S. Zientara, E. Breard, C. Sailleau, M. Beer, B. Hoffman, P.S. Mellor and P. P. C. Mertens// Virology. - 2008. - Vol. 377. - P.308-18. 5. Mertens, P.P.C. Assignment of the genome segments of bluetongue virus type 1 to the proteins which they encode/ P.P.C. Mertens, F. Brown and Sangar DV// Virology. - 1984. - Vol.135. - P.207-17. 6. Sailleau, C. Nucleotide sequence comparison of the segments S10 of the nine African horsesickness virus serotypes/ C. Sailleau, S. Moulay and S. Zientara// Arch. Virol. - 1997. - Vol.142. - P.965-78. 7. Hyatt, A.D. Release of bluetongue virus-like particles from insect cells is mediated by BTV nonstructural protein NS3/NS3a/ A.D. Hyatt, Y. Zhaoand, P. Roy// Virology. - 1993. - Vol.193. - P.592-603. 8. Development and initial evaluation of a real-time RT-PCR assay to detect bluetongue virus genome segment 1/ A.E. Shaw, P. Monaghan, H.O. Alpar, S. Anthony, K.E. Darpel, C.A. Batten, A. Guercio, G. Alimena, M. Vitale, K. Bankowska, S. Carpenter, H. Jones, C.A.L. Oura, D.P. King, H. Elliott, P.S. Mellor, P.P.C. Mertens// http://www.sciencedirect.com/science/journal/01660934. - 2007. -http://www.sciencedirect.com/science?_ob=PublicationURL&_hubEid=1-s2.0-S0166093407X03136&_cid=271147&_pubType=JL&view=c&_auth=y&_acct=C000228598&_version=1&_urlVersion=0&_userid=10&md5=3ce2e4caaf5a1dbc305c242f8e5a2345 P.115-126. 9. Bluetongue virus detection by two real-time RT-qPCRs targeting two different genomic segments/ J.F. Toussaint, C. Sailleau, E. Breard, S. Zintara, K. De Clercq// Journal of Virological Methods. - 2007. - Vol.140. - P.115-123. 10. Rapid detection and quantitation of Bluetongue virus (BTV) using a Molecular Beacon fluorescent probe assay/ G. Orr u , M. L. Ferrando, M. Meloni, M. Liciardi, G. Savini, P. De Santis// Journal of Virological Methods. - 2006. - Vol.137. - P.34-42. Test-system for detection of the 10th segment of the genome of bluetongue virus by polymerase chain reaction in real time, including seroprotection oligonucleotide primers and DNA probes, complementary to the conservative areas of the 10th segment, having the following nucleotide composition (5' to 3'):
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