80 results about "Bluetongue Viruses" patented technology

The present invention relates to an immunogenic or vaccine composition to induce an immune response or protective immune response against Orbiviruses, more specifically bluetongue virus (BTV) in an animal susceptible to BTV infection. The composition may include a pharmaceutically or veterinarily acceptable vehicle or excipient, and a vector. The vector may contain heterologous nucleic acid molecule(s), expresses in vivo in the animal BTV antigen, immunogen or epitope thereof, e.g., BTV VP2; BTV VP2 and VP5; BTV VP2 and VP5 and VP3 and / or VP7. The composition can contain an adjuvant, such as carbomer. Methods for making and using such a composition, including prime-boost regimes and including as to differential diagnosis, are also contemplated.AGACAGTGGTCAATTCCAATGGTACTGTTTGACGATAC

The invention relates to a multiplex PCR (polymerase chain reaction) primer, a probe and a gene chip for detecting the bluetongue virus, foot and mouth disease virus and bovine viral diarrhea virus. The multiplex PCR primer and probe have the nucleotide sequences shown by SEQ ID No.1 to SEQ ID and No.9. The gene chip comprises a solid-phase carrier, a sample application quality control probe, a positive hybrid quality control probe and a multiplex PCR primer for detecting the bluetongue virus, foot and mouth disease virus and bovine viral diarrhea virus and the corresponding probe. In the invention, the forward primers of three viruses are marked with fluorescence, a gene chip detection technology carrying three viruses in animal fur is established based on multiplex RT-PCR (reverse transcription-polymerase chain reaction), and the RNA virus in the fur can be sensitively and specifically detected with high flux; the three viruses are screened at the same time in detection once, and the situation that a specific method is required for each virus before is changed, thereby saving the diagnosis time, meeting the needs for quick detection of mass imported / exported fur samples of the exit-entry inspection and quarantine departments and the fur import and export enterprises, and realizing relatively high application values.

The invention discloses a duplex fluorescent RT-LAMP detection group and kit for visually identifying foot-and-mouth disease virus and Bluetongue virus and an application of the detection group and the kit. The duplex fluorescent RT-LAMP detection group comprises two groups of specific primers and probes, the first group of specific primers and probes comprise FMDV-F3, FMDV-B3, FMDV-FIB(F1c-F2), FMDV-BIP(B1c-B2) and FMDV-probes, the second group of specific primers and probes comprise BTV-F3, BTV-B3, BTV-FIB(F1c-F2), BTV-BIP(B1c-B2) and BTV-probes, and the two groups of specific primers and probes are as shown in a sequence table SEQ ID No.1 to SEQ ID No.10. The duplex fluorescent RT-LAMP detection group can identify and diagnose the foot-and-mouth disease virus and the Bluetongue virus inthe same reaction tube and has the advantages of good specificity, high sensibility, less pollution, convenience, rapidness and the like, a detection result can be directly observed by naked eyes andis judged according to colors of reaction products, and the detection group can be used for basic-level quarantine with poor conditions.

The invention discloses a gene chip and a detection method for detecting FMDV, VSV, SVDV, PPRV and BTV. The detection method comprises the step of detecting foot and mouth disease viruses (type A, Asian type I and type O), vesicular stomatitis virus, swine vesicular disease virus, Peste des petits ruminants virus and bluetongue virus. The method comprises the following specific steps: designing a PCR primer by virtue of sequence analysis of standard strain genome, and performing cloning and sequence analysis on target genes; designing a specific probe, and simultaneously detecting the foot and mouth disease viruses, vesicular stomatitis virus, swine vesicular disease virus, Peste des petits ruminants virus and bluetongue virus. The invention aims at establishing a method for detecting the foot and mouth disease viruses, vesicular stomatitis virus, swine vesicular disease virus, Peste des petits ruminants virus and bluetongue virus by adopting a microarray chip which is high in sensitivity and high in specificity and through which the time and labor are saved and the result is easily observed.

The invention discloses a multiple-connection probe amplification detection kit, primer and probe for simultaneously detecting the bluetongue viruses, the infectious bovine rhinotracheitis viruses, the bovine viral diarrhea viruses, the enzootic bovine leucosis viruses and the foot and mouth disease viruses. The multiple-connection probe is shown in sequence tables from SEQ ID NO:1 to SEQ ID NO:10. The primer is shown in sequence tables from SEQ ID NO:11 to SEQ ID NO:12. The primer, the probe and / or the multiple-connection probe amplification detection kit including the primer and the probe can detect the five vital cow disease pathogenies including the bluetongue viruses, the infectious bovine rhinotracheitis viruses, the bovine viral diarrhea viruses, the enzootic bovine leucosis viruses and the foot and mouth disease viruses at the same time, the detection time and cost are saved, and epidemic diseases can be diagnosed in time.

The invention discloses a monoclonal antibody BTV8-VP2-3E11 resistant to bluetongue virus 8 type (BTV8) VP2 protein, B-cell epitope peptide identified thereby and application, and belongs to the field of BTV8 control. A selected hybridoma cell strain capable of stably secreting a BTV8-VP2 protein resistant monoclonal antibody is stored with a microbial preservation number of CGMCC (China General Microbiological Culture Collection Center) No.7004. The experiment results show that the monoclonal antibody BTV8-VP2-3E11 secreted by the hybridoma cell strain can perform idiosyncratic reaction with the BTV8-VP2 protein but not reacts with the VP2 proteins of other serum types. The monoclonal antibody BTV8-VP2-3E11 and the BTV8-VP2 protein virus specific conserved B cell epitope peptide identified by the a monoclonal antibody can be prepared into an agent used for diagnosing BTV8 infection, thus laying a good foundation for creating serology differential diagnosis methods for BTV8 and other types of serum.

The invention discloses monoclonal antibody BTV16-3G10 resistant to bluetongue virus serum 16 type VP2 protein, B-cell epitope polypeptide identified by the monoclonal antibody BTV16-3G10, and applications of the monoclonal antibody BTV16-3G10, and belongs to the field of bluetongue prevention. The invention also relates to a hybridoma cell strain which is capable of secreting the monoclonal antibody BTV16-3G10, and monoclonal antibody secreted by the hybridoma cell strain. It is shown by indirect immunofluorescence assay that the specific reaction is observed between the monoclonal antibody and BTV16, and no cross reaction is observed between the monoclonal antibody and other serum types of bluetongue virus, Ibaraki virus or Chuzan disease virus. Further, BTV16-VP2 protein B-cell epitope polypeptide which can be identified by the monoclonal antibody is determined via cutting expression antigen short peptide and peptide scanning technology. The monoclonal antibody, and the BTV16-VP2 protein B-cell epitope polypeptide identified by the monoclonal antibody can be used for preparation of BTV16 specific differential diagnosis reagents, and foundation is provided for development of BTV epitope labeled vaccines.

The invention relates to the biotechnology field. A monoclonal antibody against a BTV VP7 protein of a bluetongue virus (BTV) of the invention is prepared by the following steps: adopting the hybridoma cell technology, taking splenocytes of BALB / c mice immunized with purified BTV for fusion with a mouse myeloma cell (SP2 / 0), after culturing the cells with an HAT selective medium, carrying out screening with indirect ELISA coated by a purified BTV antigen, a BTV VP7 protein antigen expressed by a gene engineering and a control antigen of a normal hamster kidney continuous cell (BHK21), carrying out screening and cloning by limiting dilution to obtain a hybridoma cell line which has the capacities of stable continuous culture and secretion of the monoclonal antibody (McAb) against the specific BTV VP7 protein and preparing McAb mouse ascites of the BTV VP7 protein. The monoclonal antibody against the BTV VP7 protein features strong specificity, high ascites titer, high affinity and simple preparation method, can be used in the detection method of the BTV antibody and antigen and provides an important technical means for prevention and control of bluetongue in China.

The invention discloses a biological bar code test method of bluetongue virus, taking VP7 protein of the bluetongue virus as a tested object. An MMP probe is coated with an anti-VP7 protein monoclonal antibody, and an NP probe is coated with an anti-VP7 protein polyclonal antibody. A bar code DNA chain carrying out the biological bar code test to the bluetongue virus is provided with an SEQ ID No: 1 nucleotide sequence in a sequence table, while a complementary probe NP chain is provided with an SEQ ID No: 2 nucleotide sequence in the sequence table. The invention has greater practical significance in the field of the diagnosis of the bluetongue and the inspection and quarantine of import and export, thus having wide application prospects.

The invention discloses a monoclonal antibody of anti-Bluetongue virus serum 4-type VP2 protein, a hybridoma cell strain capable of secreting the monoclonal antibody and an application of the hybridoma cell strain. According to the invention, the hybridoma cell strain which is capable of stably secreting the anti-BTV4-VP2 protein monoclonal antibody is obtained by screening positive hybridoma cells by taking VP2-4B protein, which is constructed and expressed by taking pET-28a as a vector, as an immunogen to immunize a mouse and by taking VP2-4B protein, which is constructed and expressed by taking pMAL-c5x as a vector, as an antigen, and conducting final screening. Experiments prove that the monoclonal antibody BTV4-VP2-1B6, which is secreted by the hybridoma cell strain, can participate in a specific reaction with the BTV4-VP2 protein while cannot react with other serum-type VP2 protein. The monoclonal antibody BTV4-VP2-1B6 disclosed by the invention lays a foundation for serology differential diagnosis of the BTV4 and other serum types.

The invention relates to the technical field of biology, in particular to a reagent which can simultaneously distinguish and detect four animal insect-borne diseases as well as a preparation method and application of the reagent. The animal insect-borne disease multi-RT-PCR distinguishing and detecting reagent comprises four pairs of specific primers, and the respective amplification target fragment lengths of Bluetongue virus (BTV), epizootic haemorrhagic disease virus of deer (EHDV), Vesicular stomatitis virus (VSV) and Akabane virus (AKV) are 351bp, 536bp, 300bp and 250bp. The VP7 of the BTV, the EHDV, the VSV and the AKV and a conservative fragment of an N gene are respectively selected as targets, and the primer Express software and the primer prere 5.0 software are applied to deign and combine a primer. An optimal matching and screening test and a multi-RT-PCR test are carried out on a plurality of pairs of designed primers, and four pairs of primers which can carry out the distinguishing and detection on the four animal insect-borne diseases and have high amplification efficiency and good specificity can be obtained by a plurality of reaction condition optimization and comparison tests and verification tests. A kit formed by the reagent can obtain a qualitative distinguishing and detecting result in six hours after a sample is received. The multi-RT-PCR distinguishing and detecting reagent is a sensitive and reliable method for detecting the BTV, the EHDV, the VSV and the AKV in the clinical sample.

The invention belongs to the field of virus detection, and relates to primers and a probe sequence for fluorescence RT-PCR detection for bluetongue virus serum-16. A real-time fluorescence RT-PCR detection method for BTV-16 comprises the following steps: adopting a Taqman technology, designing a group of specific primers which are only conservative in BTV-16 VP2 genes and a specific fluorescence labelling probe, and applying a fluorescence PCR instrument to detect a sample. Domestic and overseas BTV-16 viral nucleic acids can be specifically detected by using the fluorescence RT-PCR detection method established by the group of primers and the probe, and other serotypes of BTV nucleic acids cannot be detected. The method has the characteristics of being high in specificity, high in sensitivity, simple to operate, low in time consumption, high in efficiency and the like, and is capable of avoiding possible environmental pollution during a detection process.

The invention discloses monoclonal antibody BTV16-2B4 resistant to bluetongue virus serum 16 type VP2 protein, B-cell epitope identified by the monoclonal antibody BTV16-2B4, and applications of the monoclonal antibody BTV16-2B4, and belongs to the field of bluetongue prevention. The invention also relates to a hybridoma cell strain which is capable of secreting the monoclonal antibody BTV16-2B4, and monoclonal antibody secreted by the hybridoma cell strain. It is shown by indirect immunofluorescence assay that the specific reaction is observed between the monoclonal antibody and BTV16, and no cross reaction is observed between the monoclonal antibody and other serum types of bluetongue virus, Ibaraki virus or Chuzan disease virus. Further, BTV16-VP2 protein B-cell epitope polypeptide which can be identified by the monoclonal antibody is determined via cutting expression antigen short peptide and peptide scanning technology. The monoclonal antibody, and the BTV16-VP2 protein B-cell epitope polypeptide identified by the monoclonal antibody can be used for preparation of BTV16 specific differential diagnosis reagents, and foundation is provided for development of BTV epitope labeled vaccines.

The invention discloses a qualitative, quantitative detected primer and TaqMan probe of bluetongue virus, which is characterized by the following: the upstream primer of primer possesses SEQ ID No.:1 in the sequence list and downstream primer possesses SEQ ID No.:2 in the sequence list; the TaqMan probe possesses SEQ ID No.:3 or SEQ ID NO:4 DNA sequence in the sequence list; the 5'-end of probe marks report fluorescent group and 3'-end marks quenched fluorescent group; fitting for clinical and scientific study to proceed qualitative and quantitative analysis for bluetongue RNA; possessing important meaning to evaluate and observe bluetongue happening, recurrence and treating effect.

The invention discloses a monoclonal antibody BTV8-VP2-4D9 resistant to bluetongue virus 8 type (BTV8) VP2 protein, B-cell epitope peptide identified thereby and application, and belongs to the field of BTV8 control. A selected hybridoma cell strain capable of stably secreting a BTV8-VP2 protein resistant monoclonal antibody is stored with a microbial preservation number of CGMCC (China General Microbiological Culture Collection Center) No.7003. The experiment results show that the monoclonal antibody BTV8-VP2-4D9 secreted by the hybridoma cell strain can perform idiosyncratic reaction with the BTV8-VP2 protein but not reacts with the VP2 proteins of other serum types. The monoclonal antibody BTV8-VP2-4D9 and the BTV8-VP2 protein virus specific conserved B cell epitope peptide identified by the a monoclonal antibody can be prepared into an agent used for diagnosing BTV8 infection, thus laying a good foundation for creating serology differential diagnosis methods for BTV8 and other types of serum.

The invention discloses a kind of bluetongue virus (BTV) NS2 protein monoclonal antibody BTV-4D4, a B cell epitope recognized thereby and applications. BALB / c mice are subjected to totivirus immunization by utilization of bluetongue virus serotype 8 type (BTV8), splenic lymphocytes of the mice after immunization and SP2 / 0 cells are fused. A hybridoma cell strain BTV-4D4 secreting anti-BTV-NS2 protein monoclonal antibodies steadily is obtained after screening with BTV 8 as a coating antigen through an indirect ELISA method. The monoclonal antibodies secreted from the hybridoma cell strain and the BTV-NS2 protein are subjected to a specific reaction. The B cell epitope recognized by BTV-4D4 is 149RSNYDV154 after identification by utilization of a phage display technology. The monoclonal antibody and the BTV-NS2 protein B cell epitope recognized by the monoclonal antibody can be used for diagnosis or prevention of bluetongue virus infection.

GeXP multiple rapid detection primers for detection of bluetongue virus, bovine viral diarrhea virus and foot-and-mouth disease virus and a detection method are provided; the detection method for various cow diseases based on a GeXP multiple gene expression genetic analysis system is intended to be built up; the problems that when detection of the various cow diseases, a serological method has low sensitivity, conventional PCR only detects single pathogens and a gene chip method has relatively high cost are solved; three cow disease DNA positive sample standard plasmids are constructed; a reference obtained by 10-time continuous dilution of the plasmids DNA is used as a detection object, and the detection sensitivity is 10-100 copies; no cross reactions with other cow diseases happen, and no false negative results exist; the number of detected actual simples is 600, and monitoring screening requirements of various diseases of a lot of cows are met.

The invention discloses a primer combination and GeXP detection method for simultaneously identifying 5 bovine viral dermatitis viruses. The primer combination is composed of a primer pair I, a primer pair II, a primer pair III, a primer pair IV and a primer pair V. The invention also discloses a GeXP detection method for simultaneously identifying foot-and-mouth disease virus, bluetongue virus, vesicular stomatitis virus, bovine viral diarrhoea virus and infectious bovine rhinotracheitis virus. The GeXP detection method can simultaneously identify the 5 bovine viral dermatitis viruses. The method has the characteristics of higher flux, higher specificity and higher sensitivity, can be used for monitoring of bovine disease epidemiology and differential diagnosis of unexpected epidemic situations, and ensures the healthy development of cattle raising industry.

The invention discloses a multiplex RT-PCR kit for BTV-11, BTV-17, BTV-20, BTV-23 and BTV-24 genotype identification and a detection method of the multiplex RT-PCR kit. The multiplex RT-PCR kit is used for simultaneously discriminating and detecting bluetongue virus types 11, 17, 20, 23 and 24 with a single tube. According to the method, 5 pairs of PCR specific primers are designed according to conserved regions of VP2 gene sequences of various types of viruses, in addition, a pair of non-biogenic-derivation universal primers are synthesized with reference to a GeXP principle, the universal primers are separately added to upstream and downstream of each pair of specific primers to form 5 pair of specific embedded primers, reverse transcription is carried out with the specific primers, andfinally, multiplex PCR is constructed by adopting the universal primers and the specific embedded primers. By utilizing an optimized multiplex PCR system and conditions, one or more of the 5 genotypesof the bluetongue viruses can be simultaneously discriminated and detected with the single tube, specific amplification to other types of BTV, PPRV and FMDV nucleic acid is avoided, and the minimum detection concentration can reach a pg level. The method disclosed by the invention is high in sensitivity, high in specificity and easy in result observation and is timesaving and laborsaving.

The invention discloses a monoclonal antibody of anti-BTV (Bluetongue virus) NS3 protein, a B cell epitope identified by the monoclonal antibody and an application and further discloses a hybridoma cell strain which is obtained through screening and can stably secrete the monoclonal antibody. The hybridoma cell strain is named as BTV-NS3-3D8, and the microbial preservation number of BTV-NS3-3D8 is CGMCC (China General Microbiological Culture Collection Center) No.10209. The experiment proves that the monoclonal antibody secreted by the hybridoma cell strain can have the group specificity reaction with 1-24 types of NS3 protein of the BTV. Besides, the invention further discloses the specific B cell epitope, identified by the monoclonal antibody, of BTV NS3 protein. The monoclonal antibody and the specific B cell epitope, identified by the monoclonal antibody, of the BTV NS3 protein can be used for establishing a BTV antigen group identification and diagnosis method and laying the certain foundation for fundamental research of structures and functions of the NS3 protein.

The invention provides a primer and method for detecting a peste des petits ruminant virus and a bluetongue virus. According to the invention, a screened primer combination is designed, and finally itis achieved for the first time that in the same reaction tube, the peste des petits ruminant virus (PPRV) and the bluetongue virus (BTV) are simply, rapidly and accurately identified through a multiple-fluorescence RT-LAMP detection method. The multiple-fluorescence RT-LAMP primer and the method for detecting the peste des petits ruminant virus and the bluetongue virus are good in specificity, high in sensitivity and small in interference, can detect 100 mixed template copies / reactions at the minimum, and can effectively inhibit false positive results. By adopting the primer and the method, visual and accurate result judgment is achieved through differences of colors displayed by fluorescent groups, amplification can be completed only by one water bath kettle, the cost is low, the operation is convenient, the method is simple, convenient, rapid and low-cost diagnosis method, and the primer and the method are suitable for large-scale epidemiological investigation of the BTV and PPRV.

The invention aims at providing a primer and probe for detecting rift valley fever virus by applying an RPA technology. The sequence of a forward primer body is showed in SEQ ID NO:1, the sequence of a reverse primer body is showed in SEQ ID NO:2, and the sequence of a probe is showed in SEQ ID NO:3. According to the primer and probe for detecting the rift valley fever virus by applying the RPA technology, a great number of RPA primers and probes are designed according to a rift valley fever virus genomic sequence, and a primer and probe combination which can rapidly and effectively detect rift valley fever virus nucleic acid can be screened out from the great number of the RPA primers and probes. Rapid detection is conducted by utilizing the set of the primer and probe, an obvious amplification curve can be obtained by taking rift valley fever virus nucleic acid RNA in vitro transcription as a positive control, and other pathogenic nucleic acids such as foot and mouth disease virus, contagious pustular dermatitis virus, mycoplasma capricolum and bluetongue virus which serve as a template to conduct the reaction do not have the amplification curve.

The invention relates to a bluetongue virus serum 1 (BTV1) VP2 protein monoclonal antibody (BTV1-3E8), a B-cell epitope polypeptide identified by the BTV1-3E8 and application of the BTV1-3E8, belonging to the field of the prevention and treatment of bluetongue. The invention further relates to a hybridoma cell strain which secretes the monoclonal antibody. The BTV1-VP2 protein monoclonal antibody secreted by the hybridoma cell strain disclosed by the invention is named BTV1-3E8. Furthermore, the BTV1-VP2 protein B-cell epitope polypeptide, namely <465>YGQMINEMI<473>, identified by the antibody is appraised by using truncated antigen expression oligopeptides and a peptide scanning technology, and shown by sequence alignment results, the epitope polypeptide is a unique and conservative B-cell epitope polypeptide of BTV1. Shown by immunofluorescence experiment results, the BTV1-3E8 can be subjected to specific reaction with the BTV1 and does not react with BTV2 to BTV24. Thus, the monoclonal antibody BTV1-3E8 can be applied to the research and application of BTV1 specific diagnosis.

Provided are immunogenic and vaccine compositions and methods for their preparation and use, which compositions are effective in protecting against, minimizing the severity of, preventing, and / or ameliorating infection of ruminants with Bluetongue virus. Administration to an animal of at least one dose of an adjuvanted and twice inactivated Bluetongue virus composition as disclosed herein is effective in providing immunity to the animal and protection from infection with Bluetongue virus, thereby reducing the severity of and / or preventing disease caused by one or more strains or serotypes of Bluetongue virus.

The invention discloses a preparation method for a bluetongue virus core sample grain and escherichia coli for simultaneously expressing bluetongue virus vp3 and vp7 proteins. The method comprises thefollowing steps: inserting bluetongue virus vp3 and vp7 genes into a dual-expression vector pETDuet-1, thereby constructing a pronucleus dual-expression plasmid pETDuet-VP3-VP7; converting into expression type escherichia coli BL21(DE3), thereby acquiring an expression bacterium containing the pronucleus dual-expression plasmid pETDuet-VP3-VP7; performing ultrasonic breaking and sucrose gradientcentrifugation after self-induced expression, thereby acquiring the bluetongue virus core sample grain. According to the invention, high-volume high-purity expression for the bluetongue virus core sample grain with excellent immunological competence is realized in the manner of prokaryotic expression.

The invention discloses a monoclonal antibody BTV12-NS1-1F8 of anti-bluetongue-virus 12-type NS1 protein, a B cell epitope recognized by the monoclonal antibody and application of the monoclonal antibody. The monoclonal antibody is secreted by a screened hybridoma cell strain which is preserved in microbial preservation number of CGMCC No.10207; and experiments prove that the anti-BTV12 NS1 protein monoclonal antibody secreted by the hybridoma cell strain can undergo a specific reaction with BTV12-type NS1 protein. In addition, the invention also discloses a BTV12 NS1 protein specific B cell epitope recognized by the monoclonal antibody. The monoclonal antibody and the BTV12 NS1 protein specific B cell epitope recognized by the monoclonal antibody can be used for preparing reagents for identifying, diagnosing, preventing or treating BTV12 infection; meanwhile, the invention lays foundations for the establishment of an epitope based diagnosis method and the design of an epitope-labeled vaccine, and provides material reserves for further research and treatment of BTV12 type.

The invention relates to the biotechnology field. A fast-detection test trip of a bluetongue virus (BTV) comprises a sample absorption pad 1, a nitrocellulose membrane 3, an absorbent pad 6 and a baseplate 7; wherein, the sample absorption pad, the nitrocellulose membrane and the absorbent pad are fixed on the baseplate by arrangement in sequence; the sample absorption pad is a porous fiber which is linearly arranged and fixed on an impermeable material; the sample absorption pad is loaded with a colloidal gold pad which is a conjugate of a monoclonal antibody of a BTV VP7 protein and the colloidal gold; a detection line 4 is positioned on the nitrocellulose membrane and a polyclonal antibody of the BTV is coated or sprayed on the detection line; a control line 5 is positioned on the nitrocellulose membrane and a protein A is coated or sprayed on the control line. The test strip of the invention features strong specificity, high sensitivity and fast detection, can show results in 10 minutes and is especially suitable for port quarantine, field and on-site quarantine. The test strip can detect the BTV antigen directly and rapidly, without special equipments or technicists.

The invention describes the preparation of an inactivated bluetongue virus (BTV) composition, and in particular a vaccine, using an attenuated live BTV vaccine strain as masterseed. The vaccine can be administered to an animal to prevent bluetongue disease by eliciting an immune response against the bluetongue virus serotype(s) included in the composition.