69 results about "Bovine tuberculosis" patented technology

The invention discloses an immune colloidal gold indicator paper and making method to test cow tuberculosis antibody, which comprises the following parts: sample pad, connecting pad, cellulose nitrate film, absorbent pad and PVC backing liner, wherein the sample pad, connecting pad, cellulose nitrate film and absorbent pad are attached on the PVC backing liner; the connecting pad clads MPB83 protein-colloidal gold marker; the purified MPB70 protein is cladded on the cellulose nitrate film, which constitutes detecting line and quality control line formed by pure rabbit-anti-MPB83 protein lgG. The invention also provides the making method of cow mycobacterium astopic antigen MPB83, which possesses the recombinant Escherichia coli BL21 / pET28a-MPB83 in the CCTCC with reserving number at CCTCC NO:M206142.

The invention provides a bovine tuberculosis antigen specific gamma-interferon enzyme-linked immuno sorbent assay (ELISA) kit. The kit comprises a bovine gamma-interferon monoclonal antibody coated ELISA plate, enzyme-labeled bovine gamma-interferon monoclonal antibodies, and protein solution containing mycobacterium bovis MPB83 and MPB70 and mycobacterium tuberculosis ESAT6 and CFP10. The antibodies are captured and detected for different antigen surfaces of gamma-interferon respectively. A bovine tuberculosis antigen specific gamma-interferon ELISA detection method established by using the mycobacterium bovis recombinant proteins MPB83 and MPB70 and mycobacterium tuberculosis recombinant proteins ESAT6 and CFP10 is relatively stable; the specificity of the method is 96 percent, and the sensitivity of the method is 88.6 percent; and the specificity and the sensitivity of the gamma-interferon ELISA detection method for the bovine tuberculosis are greatly improved.

The invention belongs to the field of agricultural micrological gene-engineering and animal borne diseases, and relates to a spotted deer gamma-interferon double-antibody sandwich ELISA detection method, a kit thereof and application of the kit. A cell strain (CerIFN-gamma4C) capable of stably secreting a spotted deer gamma-interferon monoclonal antibody is obtained, and the storage number of the cell strain is CCTCC NO:C200966. The invention also establishes a spotted deer gamma-interferon double-sandwich ELISA detection method, which is characterized by establishing the spotted deer gamma-interferon monoclonal antibody, preparing a spotted deer gamma-interferon polyclonal antibody and the like. The kit of the invention comprises the spotted deer gamma-interferon monoclonal antibody, the spotted deer gamma-interferon polyclonal antibody, bovine tuberculosis specific three-gene fusion antigenic proteins RCE and other reagents. The invention also discloses the detection method and the application of the kit. The kit has the advantages of high specificity, high sensitivity, simple and convenient operations and quick diagnosis.

The invention belongs to the technical field of microbial genetic engineering, specifically relates to a recombinant bovine tuberculosis specific antigen protein with three fused genes and a preparation method thereof. The gene of the bovine tuberculosis specific fusion antigen protein has a nucleotide sequence and an amino acid sequence shown as in a sequence list SEQ ID NO:1. The invention discloses a recombinant Escherichia coli BL21 / pET28a-RCE capable of expressing the bovine tuberculosis specific fusion antigen protein. The Escherichia coli is conserved in China Center for Type Culture Collection (CCTCC) with a conservation number CCTCC NO:M208244. The invention further discloses a preparation method of the three gene fused antigen protein.

The invention belongs to the technical field of animal infectious disease gene engineering and discloses a bovine tuberculosis antibody detecting immune colloidal gold test strip prepared by utilizing RV3872, ESAT6 and CFP10 fusion protein, and a preparation method and application. A colloidal gold immunochromatographic test strip is established by using the fusion protein as a colloidal gold labeled antigen and a capture antigen in a detection region of a nitrocellulose membrane. The detection of the bovine tuberculosis antibody by using test strip has prominent advantages of strong specificity and high sensitivity, and simultaneously bacillus calmette-guerin immunity and nontuberculosis mycobacteria infection can be identified and detected. The test strip comprises recombinant Escherichia coli BL21 / pET28a-MPBrce, and the strain expresses mycobacterium bovis RCE proteins and is preserved in the China Center for Type Culture Collection with the collection number of CCTCC No:M208244.

The invention provides a eukaryotic expression CFP10-ESAT-6 fusion protein for diagnosis of bovine tuberculosis, which is applied to clinical diagnosis of the bovine tuberculosis. A cfp10-esat6 fusion gene is obtained by PCR (polymerase chain reaction) amplification, a recombinant plasmid pPICZ alpha A-(cfp10-esat6) is constructed, the transfer into Pichia pastoris GS115 is performed, and the high-purity CFP10-ESAT-6 fusion protein is obtained by methanol induction and affinity chromatography. The eukaryotic expression CFP10-ESAT-6 fusion protein is used for an intradermal allergic reaction which is used for diagnosis of the bovine tuberculosis and an antigen-specific bovine IFN-gamma test as a stimulus, the former has the sensitivity of 47.4% and the specificity of 83.3% in comparison with the results of a comparison allergic reaction, and the later has the positive coincidence rate of 82.8% and the negative coincidence rate of 87.4 in comparison with a bovine IFN-gamma kit, thereby showing greater value in diagnosis of the bovine tuberculosis.

The invention discloses a recombinant protein for diagnosing bovine tuberculosis, which is obtained by a method comprising the following steps: extracting the genome DNA of mycobacterium tuberculosisvar. bovis as a template of a PCR reaction to amplify out CFP10 and ESAT6 genetic fragments; using a gene splicing technique to amplify out a target gene CFP10-ESAT6 fusion gene; cloning and transforming the target gene to obtain a recombinant fungus, and inducing and expressing the recombinant fungus according to a conventional method, and purifying an expression product to obtain the recombinantprotein. The recombinant protein and a diagnostic reagent provided by the invention are used for diagnosing the bovine tuberculosis to obtain the advantages of low cost, high speed, high sensitivityand specificity.

The invention belongs to the field of immunodetection and provides a method for diagnosing bovine tuberculosis mediated by a recombinant protein mixture and a regent for diagnosing bovine tuberculosis mediated by the recombinant protein mixture; the reagent comprises the recombinant protein mixture used as specific irritant; animals infected by mycobacterium bovis can be simulated by the recombinant protein mixture so as to generate a DTH (delayed type hypersensitivity) reaction; compared with a PPD (purified protein derivative) intradermal allergic reaction test, a intradermal allergic reaction test established by regent provided by the invention has higher specificity; mycobacterium bovis infection can be distinguished from environmental mycobacteria infection; and therefore, the method and the reagent can be effectively used for clinical detection of the bovine tuberculosis.

The invention relates to a bovine cell factor electrochemical label-free impedimetric immunodetection method belonging to the technical field of electrochemical immunoassay. The method comprises the following steps: ultrasonically dispersing a nano material in a chitosan solution, thus preparing a nano material / chitosan compound; modifying a bovine cell factor antibody fixed on a glassy carbon electrode with the nano compound, thus obtaining a novel bovine cell factor immunosensor; and then, using the immunosensor for bovine cell factor electrochemical label-free impedimetric immunodetection. The detection method does not need labels, is simple, quick, low in cost, high in sensitivity and favorable in reproducibility and stability, and can be used for early diagnosis of bovine tuberculosis and research on bovine cell immune mechanism.

The invention discloses a preparation method for a bovine gamma interferon impedance type immunosensor based on zinc oxide nano-materials, and belongs to the technical field of electrochemical immunoassay. A glassy carbon electrode is modified by the zinc oxide nano-materials with different morphologies and excellent performances, and a bovine gamma interferon antibody is fixed on the electrode, so that a novel electrochemical impedance type immunosensor is prepared. The novel electrochemical impedance type immunosensor can be applied to unmarked electrochemical immunoassay of bovine gamma interferon. The immunosensor does not need to be marked, is simple, quick, low in cost, high in sensitivity, good in reproducibility and good in stability, and can be used for early diagnosing of bovine tuberculosis and research of a bovine cellular immunity mechanism.

The invention discloses a mycobacterium bovis and brucella abortus dual detection card and a preparation method thereof and aims to provide a detection card which is capable of simultaneously detecting mycobacterium bovis and brucella abortus in a bovine serum specimen, is short in detection time, high in stability, simple in operation and intuitive and reliable in result judgment and does not need other instruments or professional personnel. According to the technical key points, the detection card comprises a shell (1), wherein a sample adding hole (2) and an observation window (3) are formed in the shell (1); and a colloidal gold test strip is arranged in the shell (1). The detection card is characterized in that the colloidal gold test strip comprises a bottom plate (4); a sample pad (5), a gold label pad (6), a coating membrane (7) and an absorbent pad (8) are sequentially connected onto the bottom plate (4); a mycobacterium bovis membrane protein MPB70-MPB83 detection line T1, a brucella abortus LPS detection line T2 and a goat anti-rabbit polyclonal antibody quality control line C are formed in the coating membrane (7); and the three lines are arranged in parallel. The detection card belongs to the technical field of biology.

The invention discloses a mycoplasma bovis immune related protein, a detection kit containing the same and application thereof to detection of a mycoplasma bovis antibody. The mycoplasma bovis immunerelated protein is named p28 protein, and the amino acid sequence of the protein is shown in SEQ ID NO.2. A sensitivity test proves that the mycoplasma bovis serum antibody ELISA detection kit (MbH kit) provided by the invention can detect positive serum with the dilution multiple of 1:2560 at the minimum; and a specificity test proves that the kit has the specificity of 97.8% and has no specificreaction with the positive serums of contagious bovine pleuropneumonia (CBPP), foot-and-mouth disease (FMD), bovine tuberculosis (MB), bovine viral diarrhea (BVDV) and infectious bovine rhinotracheitis (IBRV), and has good stability and high accuracy. The invention provides a new technical means for detecting a mycoplasma bovis antibody.

The invention belongs to the field of immunodetection and provides a reagent and a method for detecting bovine mycobacterial infection. The detection reagent comprises a recombinant protein mixture used as a specific stimulation source, wherein the recombinant protein mixture can be used for stimulating animals infected with bovine mycobacteria to generate a DTH (Delayed Type Hypersensitiity) reaction and stimulating peripheral blood lymphocyte to release IFN (Interferon)-gamma. The defects of the prior art are overcome by the detection reagent, and the detection reagent has the advantages of good biosafety, low cost and feasibility in standardized production and can be effectively applied to the clinic detection of the bovine tuberculosis.

The invention provides a colloidal gold test strip detection card capable of simultaneously detecting a bovine brucellosis antibody and bovine tuberculosis gamma-interferon. The colloidal gold test strip detection card comprises a carrier plate, a sample pad, a gold label pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad and the water absorption pad are located at the two ends of the carrier plate; the nitrocellulose membrane is located in the middle of the carrier plate; the gold label pad loaded with a gold-labeled anti-gamma-interferon monoclonal antibody anda gold-labeled rabbit anti-bovine secondary antibody is arranged on the junction of the sample pad and the nitrocellulose membrane; the edge of one end of the gold label pad is overlapped under the sample pad, and the edge of the other end of the gold label pad is overlapped on the nitrocellulose membrane; a bovine tuberculosis detection line, a brucellosis antibody detection line and a quality control line are arranged from the side, close to the gold label pad, of the nitrocellulose membrane; the bovine tuberculosis detection line is coated with an anti-gamma-interferon monoclonal antibodyMT17.1, the brucellosis antibody detection line is coated with brucella lipopolysaccharide antigens, and the quality control line C is coated with goat anti-mouse IgG. The detection card can be used for effectively detecting bovine brucellosis and bovine tuberculosis.

The invention discloses a PCR detection kit for goat tuberculosis. The embodiment of the invention is a PCR detection kit for goat tuberculosis, the PCR detection kit comprises 250 [mu]L of PCR premix buffer, 150 [mu]L of ultrapure water, 50 [mu]L of Marker DL2000, 40 [mu]L of upper primer with a concentration of 10 [mu]mol / L, 40 [mu]L of lower primer with a concentration of 10 [mu]mol / L, 20 [mu]L of negative reference, and 20 [mu]L of positive reference. The primer is designed according to specific gene sections of mycobacterium bovis, and a PCR detection method for diagnosing goat tuberculosis is established by utilizing the primer. The primer is applied to the PCR rapid detection kit, so that the kit can rapidly detect the goat tuberculosis, and has a very good specificity and sensitivity. The detection result of the PCR detection kit is accurate, and the PCR detection kit has the advantages of rapidness, sensitivity, simple operation, and good using effect.

The invention discloses a protein having immunogenicity to bovine tuberculosis and application thereof. The protein having immunogenicity to bovine tuberculosis in the invention is a recombinant protein of mycobacterium BCG_2653c; and the BCG_2653c has a nucleotide sequence as represented by SEQ ID No: 1 and an amino acid sequence as represented by SEQ ID No: 2. The recombinant protein provided by the invention is applicable to a detection reagent for bovine tuberculosis.

The invention provides a device for preventing and treating cow tuberculosis by using medicine intervention. An injection mechanism is arranged on a fixing mechanism and comprises a needle cylinder, alimiting plate, a driving motor, pullback springs and an injection spring, the needle cylinder is arranged in the injection mechanism, the pullback springs are arranged on two sides of the needle cylinder, one end of each pullback spring is connected with the middle of the injection mechanism, the other end of each pullback spring is connected with the limiting plate, a guide rail is arranged atthe tail part of the needle cylinder, a lead screw is arranged at the output end of the driving motor, a sliding block is arranged on the lead screw, one end of the sliding block is arranged in the guide rail, a push rod is arranged in the needle cylinder, an injection spring and a limiting rod are arranged at the tail part of the push rod, and the other end of the injection spring is connected with the guide rail. By using the device, a medicine intervention large-scale farm is intensive, bovine tuberculosis of cattle in a high-density breeding farm is possible, the proportion of the skin difference of calves for one treatment course is reduced by 47%, the difference of positive cattle hide of the young cattle for one treatment course is reduced by 76.7%; after a course of treatment, 42%of the positive cattle hide difference is decreased, and the effect is relatively obvious.

The invention belongs to the technical field of biology, and particularly relates to an ELIS pot detection kit for detecting bovine tuberculosis and an application thereof in the field of bovine tuberculosis diagnosis. The detection kit comprises a support medium, a capture antibody and a detection antibody; the capture antibody can be produced by a hybridoma cell strain with accession number of CCTCC NO: C2017283 or a passage cell strain thereof; and the detection antibody is a gamma interferon monoclonal antibody different from the capture antibody. According to the ELIS pot detection kit for detecting bovine tuberculosis, compared with the bovine tuberculosis BOVIGAM ELISA detection kit and the bovine tuberculosis antibody detection reagent, more sensitive and high-specificity detection of mycobacterium tuberculosis infection can be realized, the false positive interference brought by the cross reaction is eliminated, and good prospects in application are shown.

The invention belongs to the technical field of animal infectious disease prevention, and relates to a mycobacterium bovis BCG vaccine low-adhesion low-invasiveness mutant strain B2909. The mutant strain contains a mutant gene of mycobacterium bovis BCG vaccine FadD18, and a nucleotide sequence thereof is shown as SEQ ID NO: 1. The mutation site is located at the 3925305 site of the genome, and islocated at the 581 site of the FadD18 gene sequence, and the gene is a novel structural gene and a functional gene. The mutant strain of the invention has low adhesion, low invasiveness, high growthrate, and no morphological difference from the wild strain. The mutant strain B2909 is preserved at the China Center for Type Culture Collection with a preservation number of CCTCC NO: M 2018866. Themutant gene and the mutant strain thereof of the present invention are expected to be applied in a mycobacterium bovis pathogenic mechanism research, an immune mechanism research and preparation of avaccine or a drug for preventing bovine tuberculosis.

The invention discloses a colloidal gold immunochromatographic test paper and a preparation and application thereof. The test paper comprises a colloidal gold labelled bovine bluetongue virus vp7 protein, a detection line and a quality control line. The test paper is suitable for detecting a bluetongue virus antibody, a preparation process is optimized through various conditions; the test paper isavailable even when bovine bluetongue standard positive serum is diluted 64 times, and has no cross reaction with bovine tuberculosis, bovine bluetongue, bovine virus diarrhea, bovine pasteurellosisand Enzootic Bovine Leukosis positive serums; and compared with an oversea commercial kit ELISA detection kit (IDEXX), coincidence rate is 98%. The test paper also has relatively better stability, cancomplete a whole detection process within 10 minutes, can quickly detect sample serum and is suitable for field fast check.

The invention discloses a bovine tuberculosis risk assessment method and model. The method comprises the following steps: determining risk factors causing bovine tuberculosis in a large-scale cattle farm; calculating a combined score weight coefficient of the risk factors through a multi-index comprehensive evaluation method; based on the combined score weight coefficient, constructing an evaluation system for evaluating the risk degree of bovine tuberculosis in the large-scale cattle farm; obtaining unqualified influence factors by using the evaluation system; and proposing a large-scale cattle farm biological safety monitoring key point. The risk degree of bovine tuberculosis in the large-scale cattle farm can be evaluated scientifically, simply, conveniently and objectively, the hidden danger of bovine tuberculosis can be found in time, and the bovine tuberculosis in the large-scale cattle farm can be early warned in time. On the basis of a large amount of infectious disease epidemic situations and related information, the multi-index comprehensive evaluation method is adopted to evaluate the risk of the bovine tuberculosis, the risk degree of the bovine tuberculosis is judged according to the evaluation value, and different prevention and control measures are implemented according to different risk levels, so that a novel, simple, practical and scientific evaluation method is provided for complex problems.

The invention discloses an immune colloidal gold indicator paper and making method to test cow tuberculosis antibody, which comprises the following parts: sample pad, connecting pad, cellulose nitrate film, absorbent pad and PVC backing liner, wherein the sample pad, connecting pad, cellulose nitrate film and absorbent pad are attached on the PVC backing liner; the connecting pad clads MPB83 protein-colloidal gold marker; the purified MPB70 protein is cladded on the cellulose nitrate film, which constitutes detecting line and quality control line formed by pure rabbit-anti-MPB83 protein lgG. The invention also provides the making method of cow mycobacterium astopic antigen MPB83, which possesses the recombinant Escherichia coli BL21 / pET28a-MPB83 in the CCTCC with reserving number at CCTCC NO:M206142.

The invention provides a bovine tuberculosis diagnosis marker and application thereof. A marker IL-8, CRP for distinguishing negative from positive of bovine tuberculosis is obtained by using a non-targeting proteomics technology for screening and a targeting proteomics technology for verification. The provided bovine tuberculosis diagnosis marker can be used to determine positive / negative state of the bovine tuberculosis as well as whether a cattle with the positive bovine tuberculosis state is in an infective period. The diagnosis marker can be used to prepare a kit or reagent for detectingthe bovine tuberculosis, provides a new detection target point for diagnosis of the bovine tuberculosis, helps timely detection and weed-out of the cattle with tuberculosis, and provides guarantee forintegrated control for the bovine tuberculosis.

The invention belongs to the technical field of infectious disease prevention and control of animals, and relates to a mycobacterium bovis bacillus calmette guerin vaccine low-invasiveness mutant strain B2801. The mutant strain contains a mutant gene of a mycobacterium bovis BCG_2658, the nucleotide sequence of the mutant gene is shown as SEQ ID NO.1, a mutation site is located behind a site 2922763 of a genome and behind a site 748 of the BCG_2658 gene sequence, and the mutant strain is a novel structural gene and a functional factor. The mutant strain has low invasiveness, low intracellularsurvival capacity, high growth speed, no cord-like structure and microcolony morphology. The mutant strain is preserved at the China Center for Type Culture Collection, with the preservation CCTCC NO:M 2018864. The seperated mutant gene and the mutant strain thereof can be expected to be applied to research on the pathogenic mechanism and immune mechanism of mycobacterium bovis and preparation ofdrugs for preventing and treating bovine tuberculosis.

The invention belongs to the field of biological detection. The invention provides a gene engineering preparation for detecting bovine mycobacterium infection. The gene engineering preparation comprises a mixture of three recombinant bovine mycobacterium proteins; the protein mixture can stimulate bovine mycobacterium infecting animals to produce a DTH (delayed type hypersensitivity) reaction. The gene engineering preparation provided by the invention has the advantages of high specificity, bio-security, component accuracy, content stability, low cost, standardized production and so on. Allergic hypodermic tests established with the detection reagents can increase experimental sensitivity and specificity, and can distinguish bovine mycobacterium infection and avian mycobacterium or non-pathogenic mycobacterium infection, so that the gene engineering preparation can be effectively used for clinic detection of bovine tuberculosis. A primer for preparing the gene engineering preparation further is provided by the invention.

The invention relates to a screening and identifying method of bovine mycobacterium immunogenicity secretion type protein. The method comprises the following steps: firstly, analysis of gene sequence of the protein coding genes of the bovine mycobacterium gene group in the gene pool is performed, and then drainage membrane helix is found among the screened protein; the protein which contains the drainage membrane helix is determined as membrane protein, and the secretory protein and the membrane protein are distinguished accordingly. The bovine mycobacterium protein with homology smaller than 36 percent with the homology of fowl mycobacterium is screened according to the principle that the bovine mycobacterium and human mycobacterium tuberculosis have higher homology; as the candidate protein, polymerase chain reaction clone is performed on the candidate protein genes, the expression vector is established and immunogenicity measurement is performed on the expression product, then bovine mycobacterium secretion type protein with immunogenicity is obtained. The probability of screening and obtaining the molecule with immunogenicity is increased greatly by the method, thus new candidate vaccine molecules are provided for prevention and cure of the bovine tuberculosis in our country.

The invention provides a bovine tuberculosis serological diagnostic marker and clinical application thereof. Serum markers, namely KLK12, MMP-1 and MMP-9, capable of identifying negative and positive bovine tuberculosis are obtained through transcriptome sequencing analysis, mouse animal experiments and clinical tests. The bovine tuberculosis serological diagnostic marker provided by the invention has the advantages that the diagnostic result of the bovine tuberculosis serological diagnostic marker is 100% consistent with that of a tubercle bacillus skin test, and the bovine tuberculosis serological diagnostic marker can identify and diagnose latent infected cattle and active tuberculosis cattle, is not influenced by infection of other mycobacteria, and has relatively strong specificity and relatively high sensitivity. The diagnostic marker provides a new target spot for diagnosis of bovine tuberculosis, can be used for preparing a serological diagnostic kit for bovine tuberculosis, does not need to collect anticoagulant and specific antigen stimulation, simplifies operation requirements, improves diagnosis efficiency, is beneficial to batch and rapid detection and elimination of tuberculosis cattle, has a wide application prospect, and provides a technical support for prevention, control and purification of bovine tuberculosis.

The invention belongs to the field of immunodetection and provides a method for diagnosing bovine tuberculosis mediated by a recombinant protein mixture and a regent for diagnosing bovine tuberculosis mediated by the recombinant protein mixture; the reagent comprises the recombinant protein mixture used as specific irritant; animals infected by mycobacterium bovis can be simulated by the recombinant protein mixture so as to generate a DTH (delayed type hypersensitivity) reaction; compared with a PPD (purified protein derivative) intradermal allergic reaction test, a intradermal allergic reaction test established by regent provided by the invention has higher specificity; mycobacterium bovis infection can be distinguished from environmental mycobacteria infection; and therefore, the method and the reagent can be effectively used for clinical detection of the bovine tuberculosis.

The invention discloses a fluorescent quantitative loop-mediated isothermal amplification (LAMP) detection method for bovine tuberculosis pathogene. The method comprises the following steps of: performing LAMP amplification on an insertion sequence IS1081 of a mycobacterium composite by internal primers shown in SEQ ID No.1 and SEQ ID No.2, external primers shown in SEQ ID No.3 and SEQ ID No.4 and loop primers shown in SEQ ID No. 5 and SEQ ID No.6 and by taking blood DNA of cattle to be detected as a template, a positive standard substance as positive control and sterile water as negative control, and collecting fluorescent signals. The method is high in sensitivity and specificity and is easy and quick to operate, and results are accurate.

The invention provides novel application of bacillus tuberculosis typus bovinus heat shock protein 65 in prevention and treatment of atherosclerosis, an administration method and an administration dosage for achieving the application. The bacillus tuberculosis typus bovinus heat shock protein 65 of the invention is obtained by means of gene engineering and biological engineering. The bacillus tuberculosis typus bovinus heat shock protein 65 has the pharmacological properties of reducing blood total cholesterol and increasing reverse cholesterol transport related gene expression.