39 results about "Bovine rotavirus" patented technology

The present invention provides a multiplex RT-PCR / PCR method, which enables in a single assay the simultaneous detection of any combination of bovine rotavirus, bovine coronavirus, Cryptosporidium parvum, and optionally, Escherichia coli strains producing K99 pili or heat-stable enterotoxin STa.

The present invention relates to a bovine rotavirus recombinant protein antigen and a preparation method thereof, in particular to a bovine rotavirus recombinant VP6 protein antigen and a preparation method thereof. The bovine rotavirus recombinant VP6 protein antigen resolves the following problems: at present, the method for diagnosing bovine rotavirus diarrhea needs a great deal of time and labor and cannot rapidly diagnose bovine rotavirus diarrhea; the process is complex; and expensive molecular biology reagents and corresponding instruments need to be used. The bovine rotavirus recombinant VP6 protein antigen is expressed by colibacillus converted by recombinant vector plasmids. The preparation method includes the following steps: (1) RCR amplification of VP6 gene; (2) acquisition of recombinant plasmids; (3) acquisition of recombinant bacteria; (4) inducible expression of recombinant VP6 protein antigen; (5) purification. The present invention provides a diagnosis antigen for the serodiagnosis of bovine rotavirus diarrhea, and the diagnosis antigen has extremely strong specificity and accuracy. The diagnosis method, which uses the bovine rotavirus recombinant VP6 protein antigen and adopts fluorescent antibody test and serological tests such as enzyme linked immunosorbent assay, has the advantages of time and labor saving and rapidness and does not need expensive reagents and instruments.

The invention relates to the field of virus detection, and particularly relates to a kit, a primer and a probe for simultaneously detecting bovine rotavirus, bovine rotavirus and bovine coronavirus. The nucleotide sequences of the primer and the probe in the kit are shown as SED ID NO: 1-SEQ ID NO: 9. The kit has the technical advantages of being easy and convenient to operate, high in specificity, high in sensitivity, good in repeatability and capable of simultaneously achieving qualitative detection and accurate quantification of BVDV, BRV and BCoV.

The invention discloses a primer, a probe and a reagent kit for RPA (recombinase polymerase amplification)-side flow chromatography detection of BRV (bovine rotavirus). The reagent kit contains primerprobe liquid for detecting the BRV through RPA technology, the forward primer sequence of the primer is as shown in SEQ ID No. 1, the reverse primer sequence is as shown in SEQ ID No. 2, and the probe sequence is as shown in SEQ ID No. 3. According to primer probe groups used in the invention, the RPA has a good amplification effect and strong strip specificity, does not have cross reaction withother viruses, has strong positive reaction in a detection area, and improves the sensitivity of detection. RPA-side flow chromatography technology is firstly adopted to establish a method for quick detection of the BRV, and by evaluation of specificity and sensitivity, the method can be used for clinical on-site detection, thereby providing a sensitive and reliable method for the on-site detection of the BRV.

Inactivated scours vaccines for immunization and protection of bovine animals from disease caused by infection with bovine rotavirus and bovine coronavirus, which comprise and effective amount of at least one inactivated viral strain are described. Polyvalent inactivated vaccines further comprising an effective amount of an antigenic component which is protective against one or more additional pathogenic organisms or viruses are also disclosed. Said vaccines are prepared from one or more strains of rota- and coronavirus, C. perfringens Type C bacteria and E. coli bacteria, and combinations thereof. Preferably, a polyvalent inactivated vaccine is provided for parenteral administration. Passive immunity is achieved in neonatal calves via immunization of pregnant cows prior to birth.

The invention discloses a bovine rotavirus, coronavirus, and viral diarrhea virus multi-connected RT-PCR detection method, which is characterized in that according to the detection method, RNA of a to-be-detected sample is taken as an RNA template, and a specificity upstream primer and a specificity downstream primer designed according to hereditary characteristics of the bovine rotavirus, coronavirus, and viral diarrhea virus are taken as a primer to perform RT-PCR proliferation to obtain a proliferation product. Then, electrophoresis detection is performed on the proliferation product with 1.5% agarose gel, and a result is recorded; according to the results of the electrophoresis detection, whether the to-be-detected sample contains the bovine rotavirus, coronavirus, and viral diarrhea virus is determined. The bovine rotavirus, coronavirus, and viral diarrhea virus detection method achieves has the beneficial effect of accurately, rapidly and efficiently distinguishing and diagnosingthe three viral disease with RT-PCR.

The invention discloses a primer combination for identifying bovine viral diarrhea disease virus (BVDV) and bovine rotavirus (BRV) and application thereof. The primer combination provided by the invention consists of single chain DNA molecules shown in sequences 1-8 in a sequence table; 5' end of a single chain DNA molecule shown in the sequence 3 is connected with fluorophore A, and 5' end of a single chain DNA molecule shown in the sequence 7 is connected with fluorophore B. According to the primer combination disclosed by the invention, the fluorophores are introduced into an LAMP method firstly, a dual-fluorescence RT-LAMP method for identifying BVDV and BRV can be established; by color observation of amplified products, two viruses can be simultaneously identified and diagnosed. The dual- RT-LAMP method built by the invention has good specificity, and can effectively amplify target genes without amplification to other pathogenic nucleic acid, so that the sensitivity is good, and 100 mixed template copy / reaction can be detected at minimum; therefore, the primer combination is a convenient, quick and low-cost diagnosis method, and is suitable for large-scale epidemiologic investigation.

The invention discloses a detection agent of bovine rotavirus and bovine coronavirus, which comprises two pairs of specific primers capable of amplifying a bovine rotavirus VP7 gene and a bovine coronavirus N gene. The invention also discloses a preparation method, which comprises the following steps of: selecting conserved sequences of the bovine rotavirus VP7 gene and the bovine coronavirus N gene; and designing and synthesizing the specific primers. The invention also discloses an application method which comprises the steps of: processing a sample; extracting RNA in an excrement sample; carrying out a jointed RT-PCR (Reverse Transcriptase Polymerase chain Reaction); and judging a result. Through the jointed RT-PCR, the detection agent can be used for rapidly and effectively detecting the BRV and the BCoV simultaneously so that the detection cost and the detection time are greatly reduced, has better specificity and sensitivity, and has the advantages of saving time and saving labor.

The present invention provides vaccine compositions for protection against human rotaviral disease designed for use in particular areas of the world. Human×bovine reassortant rotavirus comprising each of the four clinically most important VP7 serotypes of human rotavirus are combined with other VP7 serotypes typically found in the area of interest into a multivalent formulation which provides a high degree of infectivity and immunogenicity. Methods and an administration protocol for producing an immunogenic response without producing an increased risk of intussusception are also provided.

The invention discloses a yak-derived Rotavirus recombinant VP6 protein antigen and application thereof. According to the yak-derived rotavirus recombinant VP6 protein antigen and the application thereof, the nucleotide sequence of yak-derived rotavirus recombinant VP6 protein is shown in SEQ ID NO.1; the recombinant VP6 protein has good antigenicity and immunogenicity and can be efficiently expressed in a prokaryotic system; an indirect ELISA detection method is superior to a commercial detection kit, a rapid, simple, convenient and practical diagnosis method is provided for BRV antibody detection and seroepidemiology investigation in China, and the problems that an existing diagnosis method for Bovine Rotavirus is time-consuming and labor-consuming, and biological reagents and instruments are expensive can be solved; and meanwhile, a subunit vaccine has a good protection effect in mice and can be used for preventing diarrhea caused by BRV.

The invention relates to a group A bovine rotavirus detecting kit. The group A bovine rotavirus detecting kit is composed of an agent R1 and an agent R2. The invention also relates to a preparation method of the group A bovine rotavirus detecting kit. The preparation method of the group A bovine rotavirus detecting kit comprises firing colloidal gold; coupling antibody with colloidal gold nanoparticles to obtain antibody-coupled gold nanoparticle solution; performing centrifugation and settlement, and performing resuspension through buffer solution containing stabilizer and preservatives to regulate the concentration. The group A bovine rotavirus detecting kit is high in detecting sensitivity and specificity, good in stability, simple in operation, short in reaction time, applicable to a fully automatic biochemical analyzer, a special protein analyzer and a spectrophotometer, and capable of avoiding precipitates after reaction and facilitating cleaning of reaction beakers. The group Abovine rotavirus detecting kit is more reliable in detecting results compared with BRoV (bovine rotavirus) detecting data statistical analysis of samples in methods such as IPMA (immunoperoxidase mono-layer assay), ELISA (enzyme-linked immune sorbent assay), immunofluorescence and immunochromatography, and lower in production costs compared with emulsion-process production, thereby being high in marketing competitiveness.

The invention discloses a multi-nano-PCR primer group for detecting bovine rotavirus (BRV), bovine parvovirus (BPV) and a bovine viral diarrhea virus (BVDV) and application of the multi-nano-PCR primer group. The primer group contains dual-priming oligonucleotide (DPO) primer pairs used for detecting the BRV, the BPV and the BVDV correspondingly. A multi-DPO-nano-PCR detection method capable of simultaneously detecting the BRV, the BPV and the BVDV is established by combining DPO primers with a nano PCR technology. Compared with a conventional PCR method, the multi-DPO-nano-PCR detection method is time-saving and labor-saving, can quickly, accurately and specifically detect pathogens, and achieves high sensitivity on the basis of ensuring specificity. By providing the multi-nano PCR primergroup, the new method is provided for diagnosis of early infection and inapparent infection of the BRV, the BPV and the BVDV, a reliable technical means is provided for detection of clinical mixed infection of the BRV, the BPV and the BVD, and technical support is provided for epidemic disease testing, screening purification and comprehensive prevention and control.

The invention discloses a triple RPA (recombinase polymerase amplification) detection kit for a bovine viral diarrhea virus, a bovine coronavirus and a bovine rotavirus. The triple RPA detection kit comprises RPA detection primer groups for the bovine viral diarrhea virus, the bovine coronavirus and the bovine rotavirus, wherein the RPA detection primer groups are respectively composed of nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.5, SEQ ID NO.8 and SEQ ID NO.13, and SEQ ID NO.15 and SEQ ID NO.18. The kit is based on a recombinase polymerase amplification method, cDNA formed by RNA reverse transcription is used as a template for amplification reaction, and a detection result is obtained by nucleic acid gel electrophoresis. The kit can be used for simultaneously detecting the bovine viral diarrhea virus, the bovine coronavirus and the bovine rotavirus, is simple to operate, has relatively high sensitivity and specificity, and provides a rapid detection reagent for on-site screening of the bovine diarrhea virus.

The invention provides a visual multiplex fluorescent LAMP detection method which can detect bovine rotavirus (BRV) and enterotoxigenic escherichia coli (ETEC). According to the detection method, twofluorophores are introduced into an LAMP method, a result is directly judged by virtue of colors of a reaction product, for example, red is namely bovine diarrhea caused by the BRV, and green is namely bovine diarrhea caused by the ETEC. The detection method provided by the invention has the advantage that the two viruses can be identified and detected at the same time through once LAMP reaction in one reaction tube. The primer designed by the invention is high in sequence sensitivity, and each reaction can detect 100 copied mixed templates at least; and specificity is good, and a target genecan be efficiently amplified; and clinical detection effect is good. The detection method provided by the invention is convenient and rapid, does not need to use an expensive instrument, is low in cost and can realize field pathogeny detection.

The invention provides a bovine rotavirus VP8* subunit recombinant chimeric protein. According to the bovine rotavirus VP8* subunit recombinant chimeric protein, a T cell epitope polypeptide P2 in a tetanus toxin is introduced into an epitope vaccine of a bovine rotavirus VP8* subunit multi-copy chimeric gene recombinant protein, so that the immune efficacy of the epitope vaccine can be greatly enhanced, cross neutralizing immune protection can be induced, higher neutralizing antibody titer can be induced, and a high-titer anti-P[5] and P[11] genotype-specific rotavirus neutralizing antibody can be induced. The bovine rotavirus VP8* subunit recombinant chimeric protein provided by the invention can be used for preventing and controlling both G6 and G10 type rotaviruses which are mainly pandemic within a world range, and is suitable for preparing a safe and low-cost bovine rotavirus vaccine; in addition, the bovine rotavirus vaccine can be used for effectively reducing the economic loss brought by rotavirus gastroenteritis to cattle rearing by being matched with other rotavirus vaccines researched and developed at present, and has wide market prospects.

The invention provides a triple fluorescent quantitative PCR kit for simultaneously detecting a bovine rotavirus, a bovine coronavirus and a bovine viral diarrhea virus and an application, and belongs to the technical field of virus detection. The triple fluorescent quantitative PCR kit comprises three groups of specific primers and three corresponding probes aiming at the bovine rotavirus, the bovine coronavirus and the bovine viral diarrhea virus. The primer and the probe for detecting the three viruses have the advantages of strong specificity and high reaction efficiency. The triple real-time fluorescent quantitative PCR kit has the characteristics of high amplification efficiency, strong specificity, stable detection effects and good repeatability, and is suitable for dairy cow diarrhea pathogen detection, regular monitoring, dairy cow diarrhea epidemiological investigation and the like. The kit disclosed by the invention can be used for simultaneously detecting three dairy cow diarrhea pathogens in a real-time fluorescent quantitative PCR reaction system, so that the detection time is saved, the detection cost is reduced, a plurality of samples can be simultaneously detected, and the detection efficiency is high.

The invention provides a bovine rotavirus fusion protein and a calf diarrhea polyvalent vaccine, and relates to the technical field of molecular biology. The bovine rotavirus fusion protein contains aVP6 fragment, and the VP6 fragment contains an amino acid sequence as shown in SEQ ID NO.4, wherein at least one loop region in the following (a)-(c) is replaced with an epitope derived from bovine coronavirus and / or an epitope derived from escherichia coli: (a) 168th-177th amino acid residues with an amino acid sequence as shown in SEQ ID NO.1; (b) 194th-205th amino acid residues with an amino acid sequence as shown in SEQ ID NO.2; and (c) 296th-316th amino acid residues with an amino acid sequence as shown in SEQ ID NO. 3. The bovine rotavirus fusion protein contains a plurality of epitopes, and a host can generate a plurality of antibodies after the host is immunized.

The invention relates to a bovine rotavirus, a bovine coronavirus combined inactivated vaccine and a preparation method thereof. The active ingredient of the vaccine of the present invention comprisesinactivated antigen of bovine rotavirus and bovine coronavirus, and the antigenic component of the combined inactivated vaccine can be subjected to virus propagation through a continuous cell line (aiming at bovine rotavirus and bovine coronavirus) or microcarrier suspension culture (aiming at bovine rotavirus), and a seedling process and the like can be optimized. The vaccine safety, effectiveness test and antibody tracking results show that no adverse reaction is generated after immunization of cattle with the combined inactivated vaccine of the present invention, and no abortion is inducedafter immunization of pregnant cattle. Immunization of cattle produces immune protection, and newborn calves can obtain high levels of maternal antibodies through breast milk. The results show that the vaccine of the invention is safe and reliable and can be used for preventing the generation of yak diarrhea.

The invention discloses primers, probes and a kit for real-time fluorescent quantitative PCR (polymerase chain reaction) detection of bovine epidemic diseases. The kit at least comprises primers and probes for detecting BHV (Bovine herpesvirus), BPIV (bovine parainfluenza virus), SC type MmmSC (Mycoplasma mycoides subsp mycoides SC), BRSV (Bovine respiratory syncytial virus), BTV (bluetongue virus), BVDV (bovine viral diarrhea virus), BRV (bovine rotavirus), PPRV (peste des petits ruminants virus), RPV (Rinderpestvirus), VSV (vesicular stomatitis virus), FMDV (foot-and-mouth disease virus) and IC. Compared with the prior art, the four TaqMan multiple real-time fluorescent quantitative PCR systems of BHV, BPIV, MmmSC and IC, BRSV, BVDV, BTV and IC, BRV, PPRV, RPV and IC, FMDV, VSV and IC are constructed, and the specificity, repeatability and sensitivity of the systems all reach expected effects, so that the 11 pathogens can be rapidly detected at the same time.

The invention relates to the field of virus detection, and in particular relates to a kit, primer and probe for detecting bovine rotavirus. The invention provides a kit for detecting bovine rotavirus;the kit contains a nucleic acid amplification reagent; the nucleic acid amplification reagent contains a primer and a probe for detecting bovine rotavirus; the primer comprises an upstream primer asshown in SEQ ID NO:1 and a downstream primer as shown in SEQ ID NO:2; and the probe comprises a probe as shown in SEQ ID NO:3. The kit provided by the invention has the technical advantages of high sensitivity and high specificity.

The invention provides a bovine rotavirus recombinant VP7 protein antigen and a preparation method thereof and relates to a protein antigen and the preparation method thereof, and the bovine rotavirus recombinant VP7 protein antigen can be used for solving the problems that the existing bovine rotavirus detection method cannot simultaneously meet the requirements of equipment and operation simplification, good specificity and large-scale clinical popularization. The bovine rotavirus recombinant VP7 protein antigen is coded by 644bp basic groups and has a size of 40kDa. The preparation method comprises the following steps: 1, extracting genome RNA of a bovine rotavirus BRV-DQ strain; 2, carrying out reverse transcription, PCR (Polymerase Chain Reaction) amplification and purification on the genome RNA and then connecting the genome RNA with a pMD18-T carrier so as to obtain pMD18-T-VP7 plasmids; 3, simultaneously carrying out enzyme digestion on pMD18-T-VP7 and pET30a, connecting and converting into host bacteria so as to obtain recombinant bacteria pET30a-VP7 / BL21; 4, carrying out inducible expression on recombinant protein; and 5, purifying the recombinant protein so as to obtain the bovine rotavirus recombinant VP7 protein antigen. The bovine rotavirus recombinant VP7 protein antigen is applied to the field of detection of bovine rotaviruses.

The invention provides a VP7 gene-based bovine rotavirus qPCR detecting kit, comprising the components such as a specific primer of a conserved sequence of a VP7 gene, reverse transcriptase and heat-resistant DNA polymerase (Taq enzyme); the detected highest dilution degree can be up to 10<-5> to 10<-4>; the linear range of quantitative detection can be up to 10<-1> dupes / mu L; the VP7 gene based bovine rotavirus qPCR detecting kit does not have any amplification to other viral nucleic acids, bacteria and negative control groups; and the detecting kit is rapid, sensitive and specific and can be used for quantitatively detecting bovine rotavirus in various samples such as feces and blood.

The invention discloses a triple real-time fluorescent quantitative PCR (Polymerase Chain Reaction) primer and probe combination for simultaneously detecting BVDV (Bovine Viral Diarrhea Virus), BRV (Bovine Rotavirus) and BCV (Bovine Coronaviruses), the specificity of the primer and the specificity of the probe aim at the 5 'UTR gene segment of the BVDV, the VP6 gene segment of the BRV and the N gene segment of the BCV, and the nucleotide sequences of the primer and the probe are as shown in SEQ ID No.1-9. The invention further discloses a kit for detecting the BVDV, the BRV and the BCV. According to the invention, qualitative or quantitative detection of three viruses can be realized only by performing PCR amplification on a sample once, and the method has the advantages of simplicity and convenience in operation, strong specificity, high sensitivity, good repeatability and the like.

The present invention provides a bovine rotavirus VP8* subunit recombinant chimeric protein, which is a recombinant protein epitope vaccine in which T cell epitope polypeptide P2 in tetanus toxin is introduced into bovine rotavirus VP8* subunit multi-copy chimeric gene Among them, it can greatly improve the immune efficacy of epitope vaccines, and can induce cross-neutralizing immune protection, and induce higher neutralizing antibody titers, and can induce high titers of anti-P[5] and P[11] genes Type-specific rotavirus neutralizing antibodies. Utilizing the bovine rotavirus VP8* subunit recombinant chimeric protein of the present invention can simultaneously prevent and treat the G6 and G10 type rotaviruses that are mainly prevalent in the world, and is suitable for preparing a safe and low-cost bovine rotavirus vaccine. The combination of the developed rotavirus vaccine can effectively reduce the economic loss caused by rotavirus gastroenteritis to the cattle industry, and has broad market prospects.

The invention discloses a primer combination for identifying bovine viral diarrhea disease virus (BVDV) and bovine rotavirus (BRV) and application thereof. The primer combination provided by the invention consists of single chain DNA molecules shown in sequences 1-8 in a sequence table; 5' end of a single chain DNA molecule shown in the sequence 3 is connected with fluorophore A, and 5' end of a single chain DNA molecule shown in the sequence 7 is connected with fluorophore B. According to the primer combination disclosed by the invention, the fluorophores are introduced into an LAMP method firstly, a dual-fluorescence RT-LAMP method for identifying BVDV and BRV can be established; by color observation of amplified products, two viruses can be simultaneously identified and diagnosed. The dual- RT-LAMP method built by the invention has good specificity, and can effectively amplify target genes without amplification to other pathogenic nucleic acid, so that the sensitivity is good, and 100 mixed template copy / reaction can be detected at minimum; therefore, the primer combination is a convenient, quick and low-cost diagnosis method, and is suitable for large-scale epidemiologic investigation.

The invention discloses a double-antibody sandwich type ELISA (enzyme-linked immuno sorbent assay) diagnosis kit for detecting BRV (bovine rotavirus) and application thereof. The double-antibody sandwich type ELISA diagnosis kit contains a monoclonal antibody 4D10 excreted by a hybridoma cell strain with collection number of CGMCC NO.14726; futhermore, a purified rabbit-resistant anti-BRV polyclonal antibody is used as a capturing antibody, the monoclonal antibody 4D10 is used as a detection antibody, and the BRV double-antibody sandwich type ELISA method is established. Proofed by sensitivity experiment resultss, the minimum detection amount of BRV is 9.884*10<4>TCID50 / mlL; proofed by specificity experiment results, the specificity is good, and the cross reaction with PoRV (porcine rotavirus), PEDV (porcine epidemic diarrhea virus), BPV (bovine papilloma virus) and TGEV (eransmissible gastroenteritis virus) is avoided; proofed by between-run and within-run repeatability experiment results, the repeatability is good. Compared with the RT-PCR (reverse transcription-polymerase chain reaction) method, when the established double-antibody sandwich type ELISA method has a satisfactionrate of 100% iswhen being used for detecting 95 clinical samples, the satisfaction rate is 100%. The established double-antibody sandwich type ELISA method has the advantages that the specificity, sensitivity and repeatability are good; the method can be used for quickly detecting the BRV.

The invention discloses a colloidal gold immunochromatography test strip for detecting a bovine rotavirus, and a preparation method and an application thereof. The test strip includes a sample pad, acolloidal gold pad, a nitrocellulose membrane and an absorbent filter paper which are sequentially connected from left to right, and also includes a PVC base plate positioned at the bottom, wherein the PVC base plate is used to provide an assembling platform, the nitrocellulose membrane is provided with a quality control line formed by spraying a goat anti-mouse IgG and a detection line formed byspraying a rabbit anti-bovine rotavirus polyclonal antibody, the colloidal gold pad is coated with a colloidal gold particle-labeled mouse anti-bovine rotavirus VP6 protein monoclonal antibody, and the sample pad provides a position for adding a sample to be detected, wherein the mouse anti-bovine rotavirus VP6 protein monoclonal antibody is secreted by a hybridoma cell strain preserved in China General Microbiological Culture Collection Center with the preservation number being CGMCC NO.14726. The test strip prepared in the invention has the advantages of good sensitivity, high specificity, simplicity and rapidness in operation, and suitableness for onsite detection.