238 results about "Brucella" patented technology

The invention relates to a kit for recognizing a Brucella wild strain and vaccine strains A19 and S2. The kit comprises a special primer pair SEQ ID No.1 and SEQ ID No.2 for recognizing the Brucella vaccine strain A19 and a special primer pair SEQ ID No.3 and SEQ ID No.4 for recognizing the Brucella vaccine strain S2. The kit is used for conducting further sequencing analysis on a PCR amplification product after the Brucella and other conventional bacterial strains are distributed on a PCR amplification-electrophoresis detection area, and the Brucella A19 and S2 vaccine strains in a clinic sample can be rapidly and effectively recognized and diagnosed according to the sequencing result.

The present invention belongs to the field of microbe detecting technology. The oligonucleotide probe for detecting common intestinal tract pathogenic bacteria is designed on 16S rRNA and 23S rRNA of bacteria, ipaH of dysentery bacillus giant plasmid, VipR of Salmonella typhi and other gene sequence, has length of 25-50 bp, and relatively high sensitivity and specificity. The oligonucleotide probe is suitable for detection based on nucleic acid hybridization principle, especially detection based on gene chip principle. Under certain use condition, it can detect Listeria, parahemolutic vibrio, Campylobacter, etc. It may be used in many aspects, such as disease diagnosis, environment detection, food poisoning detection, etc.

The invention provides a monoclonal antibody against smooth Brucella (Brucella) lipopolysaccharides (sLPS), and preferably provides 14F4. Cross reaction tests show that the monoclonal antibody 14F4 has no cross reaction with Escherichia coli (O:157), Salmonella Dublin (C79-86) whole thallus and the LPS. On the basis, a competitive ELISA (c-ELISA) test method using the monoclonal antibody 14F4 as a competition antibody is established, and the method is suitable for testing large-scale clinical samples, and has the characteristics of being rapid, high-throughput, high-sensitivity and high-specificity.

The invention discloses a polymerase chain reaction (PCR) method for detecting Brucella based on a dual priming oligonucleotide (DPO) primer, and a DPO primer sequence thereof. The method comprises the following steps of: selecting a Brucella Omp25 gene as a target gene; through BLAST analysis of the gene sequence, selecting a conserved region of the target gene sequence, and designing and synthesizing the DPO primer, wherein the nucleotide sequence of the DPO primer is as follows: BO-DPOF: 5'-CTTTTGCTGCCGACGCCATCCIIIIIAGGAACAGC-3', and BO-DPOR: 5'-TTCGTCGTCCAAGCCGTTGTTAAIIIIIGCTTGATCT-3'; and establishing a DPO-PCR detection method so as to perform precise qualitative detection on the Brucella. The invention also relates to a detection kit which has the advantages of high detection specificity, high accuracy and high sensitivity.

The invention relates to the field of zoonosis immune diagnosis and discloses a brucellosis antibody detection immunochromatography test strip based on colloidal gold as a marking material and a preparation method of the test strip. In a quick brucellosis antibody detection technology, the 40-nm colloidal gold labeled with staphylococcus aureus protein A (SPA) is sprayed on glass fibers to form a gold labeled pad. Genes OMP31 and BP26 are cloned from a brucella genome, form prokaryotic expression recombinant plasmids and are transformed in escherichia coli to express proteins omp31 and bp26, the two proteins as coating antigens are respectively coated on a nitrocellulose membrane to serve as detection lines, and the detection lines, the gold labeled pad, a specially treated sample loading pad and water absorption paper are assembled into an immunochromatography detection device. The test strip has the characteristics of strong specificity, high sensitivity, convenience, simplicity, economy and the like, can be applied to typing detection of brucellosis antibodies of sheep and cattle, and has very important meaning and practical application value for brucellosis monitoring and prevalence control.

The invention discloses a cattle Brucella recombination strain S19-delta bp 26-BL and preparation method and application thereof. According to the invention, a cattle Brucella S19 genome DNA is used as a template, an upstream homologous arm of a bp26 genome is obtained, and a downstream homologous arm of a BLS gene, a L7/L12 gene and a bp 26 gene is obtained; the gene fragments are connected in sequence, and a fragment delta-bp 26-BL is obtained; a pRE112 and the fragment delta-bp 26-BL are separately subjected to a double digestion and are connected, and a suicide homologous recombinant plasmid is obtained; the plasmid is introduced into a competent cell of a S19-delta bp 26 bacterial strain, in the selection pressure of chloramphenicol, and a homologous recombinant single recon is obtained, and then the homologous recombinant single recon is coated on a selection medium for culture, and the cattle Brucella recombination strain S19-delta bp 26-BL is obtained. The strain has the advantages of good heredity stability, growth characteristic and appropriate virulence, and can be used as a mark vaccine strain for clinic.

The invention provides an ELISA detection kit for bovine Brucella, belonging to the technical field of protein engineering. In the kit provided by the invention, the mixed antigen of the outer membrane proteins OMP19, OMP22 and OMP28 of bovine Brucella is used as an envelope antigen of the ELISA kit; the concentration of each antigen is 1mu g/ml, and the antigens are mixed in a volume ratio of 1:1:1 to obtain the mixed antigen; the serum dilution is 1:200, and the dilution of the enzyme-labeled secondary antibody is 1:20,000; and the bovine Brucella is detected through an indirect ELISA method. The kit provided by the invention has the characteristics of high specificity, high sensitivity, high accuracy, easiness in operation and good reproducibility while the market application prospect is good.

The invention discloses crRNA, a kit and a detection method for detecting brucella. The crRNA is selected from the following sequences: crRNA1, the sequence of which is shown as SEQ ID NO. 6; crRNA2,the sequence of which is as shown in SEQ ID NO. 7; crRNA3, the sequence of which is as shown in SEQ ID NO. 8; and crRNA4, the sequence of which is as shown in SEQ ID NO. 9. According to the present invention, crRNA is designed and screened based on the Brucella Omp25 gene, meanwhile, a CRISPR-Cas12a system and an RPA isothermal amplification technology are combined, whether Brucella exists in a to-be-detected sample or not can be detected within a short time, the operation is easy, the detection speed is high, the cost is low, repeated detection can be achieved, at the same time, the detectionsensitivity and the detection specificity are remarkably improved, wherein the lowest detection limit can reach 10 copies per microliter.

The invention relates to a method for quickly identifying Brucella based on a mass spectrum technology and application thereof. The invention discloses a method for quickly identifying Brucella, which comprises the steps of PCR (polymerase chain reaction) amplification, SAP (serum alkaline phosphatase) digestion, transcription, nucleic acid enzyme cutting, purification, detection with a mass spectrometer and the like. Based on the method, a common Brucella nucleic acid fingerprint database is established. According to a peak mass spectrogram generated in experiments, Brucella in a sample to be detected can be classified and identified, and the results can be widely used in the fields of Brucella typing and classification, environmental health and public safety quarantine and the like.

The invention discloses a construction method and an expression method of a coexpression vector of L7/L12, Omp31, Rs alpha and sodC Brucella immune proteins. The disclosed coexpression vector includes a double expression vector pETDuet-1 and a double expression vector pRSFDuet-1, which are both expressed in the same host bacteria, wherein the two double expression vectors are respectively connected with encoding genes of two Brucella immune proteins. On this basis, the invention also discloses the construction method and the expression method of the coexpression vector. L7/L12, Omp31, Rs alpha and sodC Brucella immune proteins all with good immunity functions can be expressed simultaneously by the coexpression vector disclosed by the invention and are effectively used for preparing subunit vaccine of the Brucella gene engineering and preventing and controlling various brucellosis.

The invention relates to a cattle and sheep brucellosis indirect enzyme-linked immunosorbent assay antibody detection kit and a preparation method thereof, belongs to the field of detection of pathogens of zoonotic infections and aims at solving the problems of low sensitivity, poor specificity and the like in detection of Brucella by adopting other antigens. The kit is assembled by taking a recombinant Brucella BCSP31 protein with expression induced by SUMO as an expression vector in Escherichia coli to serve as an antigen-coated ELISA (enzyme-linked immunosorbent assay) plate, adding serum to be detected and rabbit anti-bovine HRP-IgG or rabbit anti-goat HRP-IgG and adding a washing buffer solution, a blocking solution, a TMB (3,3',5,5'-Tetramethylbenzidine) primer solution and a stop solution for matching. The prokaryotic expression vector, namely the SUMO, is utilized to express the Brucella BCSP31 protein in an efficient and soluble manner, the kit is used for establishing a Brucella ELISA detection method, the detection has better repeatability and specificity, and cattle and sheep Brucella can be fast and efficiently detected by utilizing the method.

The invention discloses a kit for simultaneously detecting four pathogenic bacteria Yersinia pestis, Frencisella tularensis, Burkholderia pseudomallei and Brucella by using fluorescent quantitative PCR (polymerase chain reaction) and a non-diagnostic detection method. The kit comprises specific primers and probes corresponding to the four pathogenic bacteria. The kit disclosed by the invention is convenient to use, and has the advantages of low reagent consumption, low cost, high detection specificity and high sensitivity. The detection method can detect four pathogenic bacteria for one sample, thereby greatly simplifying the operational process, reducing the repetitive operation steps, saving the time, reducing the labor consumed by repetitive operation, effectively saving the cost and implementing quick screening.

The invention relates to a therapeutic vaccine for malignant tumors and a composition thereof. The therapeutic vaccine for malignant tumors is a tumor cell line which contains plasmid of antisense nucleic acid of human transforming growth factor beta (TGF-beta2); the therapeutic vaccine composition for malignant tumors comprises the therapeutic vaccine for malignant tumors and an immunopotentiator, and the immunopotentiator is one selected from the group consisting of a Corynebacterium parvum preparation, a non-cell Corynebacterium parvum preparation, a BCG polysaccharide, a nucleic acid preparation, a Nocardia rubra-cell wall skeleton preparation, a group A Streptococcus preparation, a non-cell group A Streptococcus preparation, a Pseudomonas aeruginosa preparation, a non-cell Pseudomonas aeruginosa preparation, a Brucella preparation, a non-cell Brucella preparation, a non-cell Mycobacterium vaccae preparation and a non-cell Mycobacterium smegmatis preparation, preferably the non-cell Corynebacterium parvum preparation; and the malignant tumors include a lung cancer, a liver cancer, a pancreatic cancer, leukemia, lymphoma, an ovarian cancer, a colon cancer, a stomach cancer and a breast cancer.

The invention relates to brucella and a production method of a brucella vaccine. In the vaccine, a recombinant brucella rS2-deltaWboA strain is taken as a production strain. A smooth recombinant strain is transformed into a rough recombinant strain by deleting base sequences among WboA genes 1-897bp of a brucella S2 strain and eliminating the forming condition of an O chain in a smooth brucella cell wall LPS (Lipopolysaccharide) structure with a non-resistant gene screening technology. The safety of the rough recombinant strain is improved remarkably, and a good immune effect on brucelliasis is still kept. Due to the adoption of the strain to the production of the vaccine, the current situation that a brucella vaccine immune animal cannot be distinguished from a wild strain infected animal easily is changed, and the safety of the conventional vaccine is effectively improved.

The invention discloses a brucella omp31 antigenic epitope and a monoclonal antibody thereof, and an application of the brucella omp31 antigenic epitope and the monoclonal antibody thereof. The sequence of the epitope is GGYIGINAGYAGGKFK. The invention also discloses the monoclonal antibody identifying the epitope, and the application of the epitope and the monoclonal antibody in preparing reagents or medicines used for diagnosing, preventing or treating brucellosis.

The invention discloses a pig breed brucella bax inhibitor-1 (BI-1) gene deletion strain and a construction method and application thereof. The strain is obtained by replacing a pig breed brucella BI-1 gene with a kanamycin resistance gene, and the method includes the main steps that a pig breed brucella S2 vaccine strain genome is taken as a template, PCR amplification is conducted to obtain an upstream homologous arm and a downstream homologous arm of the BI-1 gene, the upstream homologous arm and the downstream homologous arm of the BI-1 gene and a kanamycin resistance gene fragment also obtained through PCR amplification are together cloned into a pUC18 plasmid, and a homologous recombination deletion plasmid pUC18-BI1-KanR is obtained. The plasmid is used for electrotransformation ofa pig breed brucella competent cell, and through PCR identification, an obtained positive transformant is the pig breed brucella BI-1 gene deletion strain. Compared with a wild strain, the deletion strain shows the characteristics of decreasing the growth rate, reducing colonies, increasing cell membrane permeability, enhancing cell polymerization and the like, and it is indicated that the deletion strain has the potential of serving as a brucella attenuated live vaccine.

The present invention provides multiplex PCR primer probes and a kit for detecting pet-derived zoonotic pathogens. Bartonella henselae, toxoplasma gondii and brucella belonging to pet-derived zoonoticpathogens are subjected to gene sequence analysis and comparison, triple fluorescent PCR detection primers and probes suitable for single reaction and simultaneous detection of the three pathogens are designed, and nucleotide sequences of the primers and probes are shown in SEQ ID NO.1-9, respectively. The present invention also discloses a method and a detection kit for detecting the above 3 pathogens. The detection method simplifies detection workload of 2/3 or more of the original, and shortens detection time by about 1 hour, and detection sensitivities of the bartonella henselae, toxoplasma gondii and brucella are 5, 5 and 50 copies, respectively. The multiplex PCR primer probes have no non-specific amplification of 23 common pathogens and canine and cat chromosomes, and show good specificity.

The invention relates to the technical field of microorganisms, in particular to a Brucella WXX-3 strain and application of the Brucella WXX-3 strain in degradation of fluoroquinolone antibiotics. The preservation number of the strain provided by the invention is CGMCC NO.22259. The WXX-3 strain provided by the invention can efficiently degrade fluoroquinolone antibiotics, and according to the embodiment, the highest degradation rate of ciprofloxacin reaches 44.7% after the strain is added into an LB liquid culture medium containing 20mg/L ciprofloxacin and is cultured in a dark place; besides, the strain is inoculated into livestock and poultry manure for propagation expanding for 8-10 days, then conditioning agents such as wood chips and straw are added for conventional composting for 30 days, and the degradation rate of ciprofloxacin can reach 85.7%; meanwhile, compost prepared from the strain can degrade antibiotic resistance genes, so that the safety of compost products is improved, and the strain is suitable for large-scale popularization and application.

The invention relates to a colloidal gold test paper bar for antibody detection of sheep Brucella. In the invention, lipopolysaccharide (LPS) extracted from a Brucella vaccine strain (S2) is taken as an envelope antigen of the colloidal gold test paper bar and improves the sensitivity of detection. Monoclonal-antibody-labeled colloidal gold of a mouse anti-sheep IgGFc terminal is prepared into a gold colloid pad of the colloidal gold test paper bar for antibody detection, and nonspecific adsorption of antibody protein by SPA in the tradition is changed. The monoclonal-antibody-labeled colloidal gold is taken as a colloidal gold labeled protein, so that specificity of detection is further improved. A set of serum for controlling quality of the test paper bar is prepared, and specificity and sensitivity of the test paper bar is ensured. The test paper bar can be used for on-site sampling, is convenient and rapid to use, and is quite suitable for application to a basic farm or a basic veterinarian.

The invention provides a food-borne pathogenic bacteria Raman spectrum classification model training method based on adaboost, and provides a Raman spectrum classification method based on an adaboostintegration algorithm for escherichia coli and brucella. The method comprises the following steps: performing data preprocessing on Raman spectra of two different germs; including deburring, the invention relates to a noise reduction (Savitzky-Golay filter). Sampling into numeric data; according to the method, firstly, a mesh search model is established, then PCA dimension reduction is carried outon data, then an adaboost algorithm based on a meta-classifier as a decision tree is used for calling the mesh search model to find out the most appropriate parameters, and it is verified that the adaboost algorithm of the integrated algorithm has higher classification accuracy compared with a single algorithm such as KNN and SVM.

The invention relates to a monogene deletion rough-type Brucella and a production method of a Brucella vaccine. According to the recombinant strain, base sequences between 1-897bp of the WboA gene of a Brucella 2308 strain are deleted by virtue of a non-resistance gene screening technology so as to lose the conditions for the formation of O-chain in a smooth-type Brucella cell wall LPS (Lipopolysaccharide) structure, and thus the smooth-type Brucella is transformed into a rough -type Brucella. The safety of the rough-type recombinant strain is significantly improved, but a good immune effect on brucellosis is still maintained, the recombinant bovine Brucella is named Brucella r2308-delta WboA strain (CGMCC No.8885). The current situation that Brucella vaccine immune animals are hard to distinguish from wild-strain-infected animals is changed by a vaccine produced by the strain, and the safeties of existing vaccines are effectively improved.

The invention discloses a brucella molecular marker vaccine strain for bovine species and an application thereof. The brucella molecular marker vaccine strain for bovine species, which is provided by the invention, is obtained by replacing the coding gene of a protein bp26 of brucella BH1 with the coding gene of a BLS protein and the coding gene of a protein L7/L12 and inactivating the coding gene of a wboA protein of brucella. Experiments prove that the molecular marker vaccine strain BH1deltabp26deltawboA-BL has good immunoprotection effects on brucellosis, has lower toxicity and obviously improved safety, can be used for immunoprophylaxis of brucellosis of cattle and sheep, can distinguish wild strain infected animals from immune animals by adopting the immunological technique, has great significance in monitoring, diagnosing, purifying and controlling brucellosis and has extensive application value.