44 results about "Bluetongue disease" patented technology

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 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.

This invention relates to a biological agent testing blue tongue of animals and its preparation method. The agent includes blue tongue virus VP7 gene recombination expression plasmid vectors and a blue tongue virus VPT recombination antigen got from its expression. The preparation method includes: 1, cloning BTV coding group specific antigen VP7 gene fragment to pMD18-T plasmid vector to make up of VP7 gene clone recombination plasmid, 2, sub-cloning plug pBAD / Thio TOPO expression vector, 3, converting TOP10cells, 4, screening the positive clones obtaining BTV VP7 gene segment forward plug with correct read code frame to set up BTV group specific antigen VP7 recombination expression vector 5, cultivating the vector with LB culture media containing 100mug / ml Amp.

The invention aims at providing a primer and a probe for detecting peste des petits ruminants virus by virtue of a RPA (Recombinase Polymerase Amplification) technique. According to the primer and the probe, a forward primer sequence is represented by SEQ ID NO:1, a reverse primer sequence is represented by SEQ ID NO:2, and a probe sequence is represented by SEQ ID NO:3. According to a method, a large number of primers and probes are designed according to genomic sequences of the peste des petits ruminants virus, and a pair of primer and probe combinations capable of rapidly detecting nucleic acid of the peste des petits ruminants virus are screened out; obvious amplification curves can be obtained by carrying out rapid detection by virtue of the primer and the probe and taking nucleic acid RNA of the peste des petits ruminants virus of domestically separated Tibet strains and Xinjiang strains as a template, and no amplification curve is obtained through a reaction by taking other pathogenic nucleic acids such as foot and mouth disease virus, contagious pustular dermatitis virus, mycoplasma capricolum and bluetongue disease virus as the template.

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 an anti-sheep virus disease yolk antibody feed additive, an injection and a preparation method thereof. The anti-sheep virus disease yolk antibody feed additive and the injection respectively are extracted and prepared by the collected yolk antibody of the immunized laying hens which are immunized by the single vaccine which is prepared form sheep pustule virus, sheep bluetongue disease virus and sheep foot and mouth disease virus or the combined vaccine which is prepared from sheep pustule virus and / or sheep bluetongue disease virus and / or sheep foot and mouth disease virus. The anti-sheep virus disease yolk antibody prevents virus diseases through improving the antibody level of sheep antibodies on corresponding pathogen microorganisms causing the virus diseases. The feed additive of the invention is convenient to be fed to sheep with obvious control effect. The anti-sheep virus disease egg yolk antibody injection is provided with faster efficacy in the onset of the sheep. The anti-sheep virus disease yolk antibody feed additive, the injection and the preparing method thereof has the advantages of obvious efficacy, low cost, fast effect, no side effects, no drug-resistant strain which can be produced, simple preparation, wide raw material resource, convenient use and easy application.

The invention discloses a functional feed for treating bovine bluetongue and a preparation method thereof. The functional feed comprises the following raw materials: corns, corn straws, soybean meal, cotton seed meal, wheat bran, table salt, alfalfa hay, sugarbeet, urea, calcium hydrophosphate, mountain flour, sorghum, potatoes, fish meal, enzymic preparation, an amino acid additive, a vitamin additive and a microelement additive. The preparation method comprises the following steps: crushing raw materials, mixing the raw materials, preparing feed granules and drying the feed. The functional feed and the preparation method have the beneficial effects that the feed contains the traditional Chinese medicine additives which have the functions of removing pathogenic heat to cool blood, detoxifying and astringing sore, has the effects of strengthening the body resistance to eliminate pathogenic factors, and has the advantages of relatively good curative effect, no drug residue, low cost and the like; the traditional Chinese medicine additives and other feed raw materials are mutually matched and interact with each other, so that the functional feed can be used for treating the bovine bluetongue and also can meet the nutritional demands of bovine.

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 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.

The invention discloses a primer used for blue tongue virus qualitative and quantitative measurement. The upstream primer of the primer is provided with SEQ ID NO. 1 in the sequence table and the downstream primer of the primer is provided with SEQ ID NO. 2 in the sequence table. The primer can be used for qualitative and quantitative analyzing of the blue tongue virus ribonucleic acid of the animals which is infected with the blue tongue virus in clinic and scientific researches, and the primer is provided with significant meaning to the judgment of occurring and recurring of the blue tongue virus, the curing effect evaluation and the dynamic observation of the patients condition; the invention can play an important role in the animal medicine detecting field.

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 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.

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 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.

The invention discloses an African swine fever virus (ASFV) P54 antibody detection method and kit based on bluetongue virus core-like particles with a chimeric ASFV P54 protein antigen epitope. The chimeric core-like particle is used as a coating antigen, the antigen epitope is better presented in a form similar to that of an original virus, and meanwhile, the monoclonal antibody with the specificity of the antigen epitope is used as a blocking enzyme-labeled antibody, so that better specificity and better sensitivity are achieved. The ASFV P54 antibody detection kit is high in safety, good in sensitivity, high in specificity, high in accuracy and convenient to operate, and compared with an imported kit, the detection coincidence rate reaches 100%.

The invention relates to a sequence optimized BT (bluetongue) virus nonstructural protein NS3 gene, a corresponding expression vector, a host cell and a preparation method of nonstructural protein NS3. The NS3 gene is optimized, a cold shock prokaryotic recombinant expression vector pCold-NS3 is constructed, expressed Escherichia coli BL21 (DE3) is transformed, and the soluble expressed BT virus nonstructural protein NS3 is obtained by inducible expression. The NS3 gene is optimized and expressed for a long time under a low temperature condition by the cold shock expression vector and a self-induced expression medium, and high soluble expression of the BT nonstructural protein NS3 is realized.

The invention discloses an anti-bluetongue antibody feed additive and a preparation method thereof. The feed additive is prepared by immunizing non-immunized laying hens with a bluetongue virus vaccine, collecting yolks of the immunized laying hens, extracting and preparing. The anti-bluetongue antibody prevents and cures bluetongue by improving the antibody level of a goat body to the pathogenic microorganism causing the viral disease. Goats can be feed with the feed additive conveniently, and preventive and curative effects are significant. The feed additive has the advantages of significant curative effects, low cost, quick effectiveness, no toxic or side effects, no generation of drug-resistant strains, simple preparation, widely available raw materials, convenience in use and easy popularization.

The invention discloses a multi-RT (reverse transcription)-PCR (polymerase chain reaction) reagent kit for genotyping identification of BTV (bluetongue virus) genotypes 2, 3, 4, 7 and 12 and a detection method of the BTV genotypes 2, 3, 4, 7 and 12, which are used for simultaneous identification and detection of the BTV genotypes 2, 3, 4, 7 and 12 through a single tube. According to different genotype virus VP2 gene sequence conservation areas of bluetongue, 5 pairs of PCR specific primers are designed in the method, a pair of non-biologically derived universal primers are synthesized by utilizing a GeXP principle, the universal primers are respectively added to the upstream and downstream of each pair of specific primers to form 5 pairs of specific chimeric primers, the specific primers are used for performing inverse transcription, and multi-PCR is constructed by combining the universal primers with the specific chimeric primers. By utilizing an optimized multi-PCR system and condition, one or more types of 5 genotypes, i.e., BTV genotypes 2, 3, 4, 7 and 12 can be simultaneously identified and detected through the single tube, other types of PTV, PPRV (peste des petits ruminantsvirus) and FMDV (foot-and-mouth disease virus) nucleic acids are not subjected to specific amplification, and the lowest detection concentration can reach a pg level. According to the method constructed by the invention, the sensitivity is high, the specificity is strong, the time and labor are saved, and the result is easy to observe.

The invention relates to the technical field of veterinary drugs, in particular to an oil for treating animal pustular dermatitis and a preparation method thereof. The oil for treating animal pustulardermatitis is prepared from the following raw material formula: 10-20ml gentamicin, 50-100ml 10% iodine, 3-6g cool oil, 8-10 million units of vitamin A, and 500-1000ml peanut oil. The oil can be usedfor treating pustular dermatitis of animal skin such as sheep infectious pustulosis, sheep pox, bluetongue disease, necrotizing bacillus disease and the like, has an obvious curative effect, is shortin treatment course, high cure rate, can effectively alleviate pain of diseased animal, and can effectively prevent human body from contacting infection.

The invention discloses a bluetongue virus-8 detection reagent, detection method and application. The reagent comprises a specific primer pair and a probe, sequences of forward and reverse primers of the primer pair are shown as Seq ID NO.1 and 2, and a sequence of the probe is shown as Seq ID NO.3. In the sequence shown as Seq ID NO.3, fluorescence quenching group-dT is modified by a 32th basic group, a 33th basic group is replaced by a basic group analogue, a fluorescence group-dT is modified by a 35th basic group, and C3Spacer is modified by a 3' terminal. By one-step reverse transcription recombinase polymerase amplification, sensitive, specific and efficient bluetongue virus-8 detection can be realized. By adoption of the reagent, convenient onsite bluetongue virus-8 detection can be realized, and quickness in acquisition of detection results is achieved. An important significance to quick prevention and control of bluetongue virus-8, prevention of epidemic spread and maximum guarantee of production safety is achieved.

The invention discloses an RT-PCR kit and method for identifying the serotype of BTV (Bluetongue virus, BTV), and belongs to the technical field of veterinary infectious disease detection. The RT-PCRkit is mainly used for serotype identification of the BTV of 12 serotypes, and the 12 serotypes are BTV-1, BTV-2, BTV-3, BTV-4, BTV-5, BTV-7, BTV-9, BTV-12, BTV-15, BTV-16, BTV-21 and BTV-24 respectively. The RT-PCR kit comprises specific RT-PCR amplification primers of 12 serotypes of BTV, and the nucleotide sequences of the specific RT-PCR amplification primers are as shown in SEQ ID No. 1 to SEQ ID No. 24. The kit has the advantages of being good in specificity, rapid, simple, convenient and low in cost, accurate typing of the 12 BTV serotypes can be conducted, the genetic characteristics of a BTV gene segment 2 can be obtained through sequencing of the amplification products, and data is provided for analysis of evolutionary characteristics of viruses.

A multiplex-PCR (polymerase chain reaction) kit for detecting six viruses of sheep and goats simultaneously comprises six pairs of specific primers, wherein the primers used for detection of the bluetongue virus are BTV-F and BTV-R respectively; the primers for detection of the foot and mouth disease virus are FMDV-F and FMDV-R respectively; the primers for detection of the peste des petits ruminants virus are PPRV-F and PPRV-R respectively; the primers for detection of the sheep pox virus are SPPV-F and SPPV-R respectively; the primers for detection of the goat pox virus are GTPV-F and GTPV-R respectively; the primers for detection of the sore mouth disease virus are ORFV-F and ORFV-R respectively. The sequences of the primers are nucleotide sequences shown in SEQ ID NO: 1-12. The multiplex-PCR kit can be used for detecting nucleic acid of the six viruses including the bluetongue virus, the foot and mouth disease virus, the peste des petits ruminants virus, the sheep pox virus, the goat pox virus and the sore mouth disease virus in sheep and goat clinical samples and has the characteristics of high sensitivity, high specificity, simple operation and reduction of detection time.

The invention discloses an RTqPCR (Reverse Transcription Quantitative Polymerase Chain Reaction) kit for identifying a BTV (Bluetongue Virus) serotype, and belongs to the technical field of veterinaryinfectious disease detection. The RTqPCR kit is mainly used for identifying the serotypes of 12 serotypes of bluetongue viruses, and the 12 serotypes of bluetongue viruses are BTV-1, BTV-2, BTV-3, BTV-4, BTV-5, BTV-7, BTV-9, BTV-12, BTV-15, BTV-16, BTV-21 and BTV-24 respectively. The RT-qPCR kit comprises a primer pair combination and a corresponding probe combination, wherein the primer pair combination is used for specifically amplifying Seg-2 gene sequences of BTV strains of 12 serotypes. The detection kit and method provided by the invention have the characteristics of high sensitivity, strong specificity, high detection flux and simple, convenient and rapid operation, and can be used for identifying the BTV serotype so as to realize accurate identification of the BTV serotype in a temporary sample.

The invention provides a primer and a method for detecting Peste des petits ruminants virus and bluetongue virus. The primer combination designed and screened by the present invention finally successfully realizes the simple, rapid and accurate identification of Peste des petits ruminants virus (PPRV) and bluetongue virus in the same reaction tube for the first time through multiple fluorescent RT-LAMP detection methods (BTV). The multiple fluorescent RT-LAMP primers and method for detecting Peste des petits ruminants virus (PPRV) and bluetongue virus (BTV) provided by the present invention have good specificity, high sensitivity and low interference, and can detect at least 100 Mix template copies / reactions and effectively suppress false positive results. The primers and the method provided by the present invention can intuitively and accurately judge the results of the different colors displayed by the fluorescent groups, and only need a water bath to complete the amplification. The cost is low and the operation is convenient. It is a simple, fast, low-cost A cost-effective diagnostic method for large-scale epidemiological investigations of BTV and PPRV.

The invention discloses a feed additive for preventing and treating ovine bluetongue. The feed additive is prepared from the following raw materials in parts by weight: 10-15 parts of radix clematidis, 10-15 parts of kadsura pepper stems, 8-10 parts of caulis sinomenii, 8-10 parts of cortex acanthopanacis, 12-14 parts of Chinese starjasmine stems, 3-5 parts of ferula asafetida, 3-5 parts of resina toxicodendri, 4-6 parts of purslane, 4-6 parts of guava, 2-3 parts of scandent hop, 2-4 parts of lonicerae flos, 1-6 parts of quispualis indica, 12-16 parts of pumpkin seeds, 16-18 parts of pomegranate barks, 16-18 parts of orobanche coerulescens, 9-12 parts of periostracum cicada, 3-6 parts of asarum, 3-6 parts of chrysanthemum, 2-6 parts of horsetail, 1-4 parts of polygonum aviculare, 2-5 parts of lacca and 20-30 parts of common andrographis herb. The feed additive disclosed by the invention has the benefits that the feed additive disclosed by the invention is overall prepared from traditional Chinese medicinal materials, so that the feed additive is less in toxic action, low in residual amount, obvious in preventing and treating effect on industry of animal husbandry and good in market prospect.