39 results about "African Swine Fever Virus Infection" patented technology

The invention discloses a fluorescent quantitative PCR (Polymerase Chain Reaction) detection reagent, a kit and a detection method for an African swine fever virus (ASFV). The detection reagent is targeted to a conserved segment of an ASFV VP72 gene, and comprises a pair of specific primers, a specific probe and an internal labeling probe. The kit comprises a PCR mixed solution, Taq DNA polymerase, DEPC (diethylpyrocarbonate)-H2O, an ASFV positive quality product, an ASFV negative quality product, a quantification standard substance and a pseudovirus internal labeling solution, wherein the PCR mixed solution comprises a pair of specific primers, a specific probe and an internal labeling probe. The detection method comprises the steps of extraction of total DNAs, preparation of reaction components, making of a standard curve by diluting the standard substance, amplifying and result judgment. The reagent and the kit have the advantages of high specificity, high sensitivity, no pollution and high flux, and can be used for accurately detecting infection, inapparent infection or continuous carrying of a toxic host of low-content ASFV. The detection method is quick, specific and sensitive, and can be used for quantitatively detecting a large quantity of samples at the same time.

The invention discloses an RPA (recombinase polymerase amplification) primer and a detection kit for rapidly detecting African swine fever viruses. Target genes can be effectively amplified, specificity is 100%, detection sensitivity is 102 copy / reaction, and sensitivity is equivalent to that of fluorescent quantitative PCR (polymerase chain reaction). Cross reaction between the RPA amplificationprimer and one of classical swine fever viruses, vesicular exanthema swine viruses I, porcine reproductive and respiratory syndrome viruses, porcine circoviruses and the like is omitted. A RPA isothermal amplification system is rapidness in reaction and wide in temperature range, effective amplification of the target genes can be achieved at the temperature of 38-46 DEG C, and the detection kit can rapidly, efficiently and sensitively detect the African swine fever viruses and has the advantages that the kit is simple to operate, high in specificity, safe and free from pollution, reaction results are easily observed and the like. Effective technical means are provided for on-site rapidness detecting and screening of infection nucleic acid of the African swine fever viruses, and the RPA amplification primer has great significance for control of infection spreading of the African swine fever viruses in China and inspection and quarantine in infected areas and entry and exit ports.

The invention discloses an African swine fever virus antigen duplex detection reagent and a preparation method thereof. The African swine fever virus antigen duplex detection reagent comprises a PVC bottom plate, wherein a sample pad, a colloidal gold marking pad, a nitrocellulose membrane detection pad and a water absorption pad are arranged at the top of the PVC bottom plate from left to right in sequence. According to the African swine fever virus antigen duplex detection reagent disclosed by the invention, by means of the detection of main and fairly conserved antigenic two proteins (p30 and p72) of AFSV, two results can be simultaneously obtained by once sample injection, furthermore, AFSV infection can be prompted as long as any one of the two results is positive, so that the detection rate is improved, whether early pig fever is caused by African swine fever virus infection can be quickly detected so as to discover the epidemic situation and take effective prevention and controlmeasures, furthermore, the African swine fever virus antigen duplex detection reagent has high sensitivity, high specificity, high speed (5-10min) ), simple and convenient operation, good repeatability, stable structure, good use performance, and is suitable for farmers to carry out self-examination or for professional institutions to carry out investigation of epidemic situation.

The invention provides a fluorescent quantitative PCR detection reagent, detection kit and detection method for identifying and diagnosing infection and immunization of African swine fever, and belongs to the technical field of biology. Specific primers and probes are designed aiming at CD2v of the African swine fever and eGFP of a fluorescent protein. An ASFVP72 / CD2v dual fluorescent PCR detection method or ASFVP72 / CD2v / eGFP triple fluorescent PCR detection method formed through combination with a P72 gene can be used for detecting the infection of the African swine fever and identifying a wild strain of a African swine fever virus and a CD2v gene deletion type recombinant vaccine strain, and thus, infected animals and immunized animals can be differentiated. The fluorescent PCR detectionkit has the advantages of simplicity and convenience in operation, high sensitivity, strong specificity, good repeatability and the like, and an important monitoring measure is provided for epidemicsituation detection and disease prevention and control.

The invention provides an African swine fever prevention and / or treatment neutralizing antibody which is an embedded monoclonal antibody and comprises a heavy chain and a light chain. A light chain variable region has a sequence shown as SEQ ID No:6, a heavy chain variable region has a sequence shown as SEQ ID No:2, and both a light chain constant region and a heavy chain constant region come froma swine derived antibody. The invention further provides a gene coding the embedded monoclonal antibody and an expression vector or a transformant comprising the coding gene. The expression carrier can be obtained by inserting the coding gene into pAAV-CAG. The invention further provides a method of utilizing the expression vector to prepare AAV. The antibody sequences or the transformant can beused for preparing detection reagents for African swine fever virus, agents for inducing immunoreaction aiming at African swine fever virus antigen in tested animals or agents for preventing animals from being infected by the African swine fever virus, and rapid and lasting antibody protection such as African swine fever virus vaccines can be provided.

The invention relates to the technical field of biological medicine, in particular to an anti-African swine fever virus and anti-CD double-target pig-derived antibody, a preparing method and application. The prepared anti-African swine fever virus and anti-CD pig-derived double-target compound antibody has two antigen combination sites which are combined with African swine fever viruses and immunecells respectively to generate the bridging effect and realize the two major functions; on one hand, the antigen proteins which cause immunosuppression and immune escape are inactivated, the immunosuppression and immune escape are eliminated, and the African swine fever viruses entering a body are killed; on the other hand, the functions of T cells are activated, the immune system is restored, the cell-mediated immunity is started, and virus expansion is prevented; meanwhile, infected cells are split and dissolved, phagocytes are activated to clear away the viruses and the infected cells, andthe aim of preventing and treating the African swine fever viruses is achieved. By means of the compound antibody, a biological medicine and a biological safety feed additive which eliminate the infection of the African swine fever viruses and prevent and treat the African swine fever can be prepared.

The invention provides a PCR primer and probe for detecting infectivity of an African swine fever virus, a kit and a detection method. Primer sequences are shown in SEQ ID No. 1~2, and a probe sequence is shown in SEQ ID No. 3. The kit provided by the invention contains the PCR primer and probe and a nucleic acid dye, which is bonded to nucleic acid and has photosensitive characteristics; and whether the infective African swine fever virus is present in a sample to be detected or not is rapidly judged through pretreating the sample to be detected with the photosensitive nucleic acid dye, taking a sample free of photosensitive dye pretreatment as a control sample, then, extracting nucleic acids from the sample to be detected and the control sample, detecting African swine fever virus conserved gene copy numbers of the sample to be detected and the control sample separately by a qPCR method, and comparing the gene copy numbers of the sample to be detected and the control sample. The kitis short in detection consumed time, high in specificity, reliable in result and simple in result judgment, is applicable to any laboratory, prevention and control units and veterinary stations of alllevels of grass roots, large-, medium- and small-size farms and the like and is of far reaching importance in judgment on measures such as killing, prevention and control on the African swine fever virus.

The invention provides an African swine fever virus antibody ELISA (Enzyme Linked Immuno Sorbent Assay) detection kit. A support medium of the kit is coated with an African swine fever virus antigen,and the African swine fever virus antigen is African swine fever virus p30 and / or protein delta p54 protein. The African swine fever virus p30 protein is as shown in SEQ ID No. 1, and the African swine fever virus delta p54 protein is as shown in SEQ ID No. 2. The African swine fever virus antibody ELISA detection kit disclosed by the invention is good in specificity, sensitivity and repeatability, the sensitivity of the kit coated with the two antigens is equivalent to that of indirect immunofluorescence detection, antibody positive can be detected earlier at a low antibody level in the earlystage, a basis is provided for clinical swine herd infection conditions, and the kit plays an important role in preventing and controlling African swine fever virus infection.

The invention provides an African swine fever virus vaccine, and a preparation method and application thereof. The preparation method of the African swine fever virus vaccine comprises the following steps of: providing an AAV vector containing a recombinant porcine interferon coding gene; providing an AAV vector containing a recombinant African swine fever virus neutralizing antibody coding gene;providing an AAV vector containing coding genes of multiple antigen proteins of the recombinant African swine fever virus; providing an AAV vector containing a gene capable of transcribing shRNA for akey gene of the African swine fever virus; and transfecting host cells by using the AAV vectors respectively, and performing culture and post-treatment to obtain a plurality of recombinant AAV viruses. The African swine fever virus vaccine provided by the invention can provide multiple barriers for preventing and treating ASFV virus infection, so that comprehensive prevention and treatment of theAfrican swine fever virus infection can be realized, and the African swine fever virus vaccine has the advantages of high safety, strong immunogenicity, no pathogenicity to animals and the like.

The invention discloses a fluorescent quantitative PCR (Polymerase Chain Reaction) detection reagent, a kit and a detection method for an African swine fever virus (ASFV). The detection reagent is targeted to a conserved segment of an ASFV VP72 gene, and comprises a pair of specific primers, a specific probe and an internal labeling probe. The kit comprises a PCR mixed solution, Taq DNA polymerase, DEPC (diethylpyrocarbonate)-H2O, an ASFV positive quality product, an ASFV negative quality product, a quantification standard substance and a pseudovirus internal labeling solution, wherein the PCR mixed solution comprises a pair of specific primers, a specific probe and an internal labeling probe. The detection method comprises the steps of extraction of total DNAs, preparation of reaction components, making of a standard curve by diluting the standard substance, amplifying and result judgment. The reagent and the kit have the advantages of high specificity, high sensitivity, no pollution and high flux, and can be used for accurately detecting infection, inapparent infection or continuous carrying of a toxic host of low-content ASFV. The detection method is quick, specific and sensitive, and can be used for quantitatively detecting a large quantity of samples at the same time.

The invention relates to a real-time fluorescent PCR amplification primer group for identifying and testing African swine fever viruses, the amplification primer group consists of upstream and downstream primers and probe primers of an African swine fever virus p72 gene, a CD2v gene and an MGF360-14L gene, and the invention also relates to a kit containing the real-time fluorescent PCR amplification primer group for identifying and testing the African swine fever viruses. By utilizing the high sensitivity of the primer group, wild strain infection or gene deletion vaccine strain immunization can be distinguished, the African swine fever virus infection can be prevented and controlled as soon as possible, and meanwhile, the kit can accurately detect suspected infection samples from different regional sources.

The invention belongs to the technical field of livestock disease detection and particularly relates to a method for detecting whether African swine fever virus carried in soil of a pig farm or not. A detection process of the method includes steps: culturing earthworms in soil around a pig house, or a production sewage area, or a position for treatment of pigs dying from diseases or an access of the pig farm, and then detecting whether the African swine fever virus is carried inside and outside earthworm bodies or not. The method is high in detection efficiency and accuracy, and risks of the farm can be anticipated, so that loss reduction is realized; aiming at the pig farm in an area with African swine fever virus infection, an important basis can be provided for production recovery in the pig farm to reduce the risks of re-infection after production recovery; in addition, a scientific basis is provided for daily disinfection management and the like in the pig farm.

The invention belongs to the technical field of African swine fever treatment, and particularly relates to new application of potassium iodide or a composition containing the potassium iodide to prevention or treatment of African swine fever. The invention accidentally finds that potassium iodide (KI) can inhibit the expression of African swine fever virus proteins p72 and p30, inhibit the copy number and virus titer of African swine fever virus genes and inhibit the replication and infection of ASFV, and is used for preventing or treating African swine fever; meanwhile, it is found that potassium iodide (KI) and ML-60218 are combined to remarkably inhibit expression of African swine fever virus proteins p30 and p72, viruses are prevented from invading host cells, and the potassium iodide (KI) and ML-60218 can be used for inhibiting the whole period of ASFV infection; and potassium iodide (KI) and ML-60218 are combined to obviously inhibit the replication of the African swine fever virus and reduce the virus titer after infection of the African swine fever virus, so that the ML-60218 can be used as an inhibitor of the African swine fever virus and is used for preventing or treating the African swine fever.

The present invention discloses a PCR primer group, a kit and a detection method for detecting an African swine fever virus MGF 360-505R gene. The primer group comprises primers and a probe with nucleotide sequence shown as SEQ ID NO.1-3; the kit comprises the primers, the probe and a reaction reagent. When the kit is used, an S-type amplification curve appearing on an ROX channel is positive to the African swine fever virus, a non-S-type amplification curve is negative to the African swine fever virus infection, so that the kit can be expected to be used for differential diagnosis of a deletion strain. The provided kit can effectively prevent aerosol pollution, and is also simple to operate, low in cost, high in sensitivity and good in specificity, and suitable for export quarantine, foodsanitation and African swine fever virus identification and detection in livestock farms.

The invention discloses an immortalized porcine macrophage cell strain as well as a construction method and an application thereof, and belongs to the technical field of bioengineering. The immortalized porcine macrophage cell strain provided by the invention is obtained by co-transfecting immortalized genes SV40LT and hTERT into primary porcine bone marrow-derived macrophages in a lentivirus infection manner, the provided immortalized porcine macrophage cell strain is suitable for culturing African swine fever virus, can obtain high virus titer, is reliable and stable, can be continuously passed, and can be effectively applied to immunological research in the aspect of resisting African swine fever virus infection and production of African swine fever virus vaccines.

The invention discloses a pharmaceutical composition capable of preventing African swine fever as well as injection and application thereof. The pharmaceutical composition capable of preventing African swine fever is prepared from the following raw materials in parts by weight: 10-1000 parts of gypsum, 5-200 parts of common anemarrhena rhizome, 5-200 parts of lophatherum herb, 5-200 parts of raw radix rehmanniae, 5-200 parts of radix scrophulariae, 5-200 parts of rhizoma coptidis, 5-200 parts of baical skullcap roots, and 5-200 parts of fructus gardeniae. The raw materials are extracted by water or alcohol so as to obtain a drug extract; the drug extract is dissolved in water; and then, benzenemethanol, tween-80 and activated carbon injection are added, and filtration is carried out so asto obtain the pharmaceutical composition capable of preventing African swine fever. The pharmaceutical composition disclosed by the invention has preventive effects on a plurality of viral infectionssuch as African swine fever virus infection and the like; and moreover, the pharmaceutical composition is free of antibiotics and drug residues. In addition, the pharmaceutical composition capable ofpreventing African swine fever has significant preventive effects on African swine fever virus that the effective rate is about 80%.

The invention belongs to the technical field of African swine fever treatment, and particularly relates to novel application of ML-60218 in preventing or treating African swine fever. It is accidentally finded that ML-60218 can remarkably inhibit the expression of African swine fever virus protein p30 and prevent viruses from invading host cells, and can be used for inhibiting early infection of ASFV; and the ML-60218 can obviously inhibit the replication of the African swine fever virus and reduce the virus titer after infection of the African swine fever virus, and can be used as an inhibitor of the African swine fever virus for preventing or treating the African swine fever.

The present invention discloses a class of shRNAs used for inhibiting replication of African swine fever viruses. The shRNAs contain two shRNA sequences such as SEQ ID NO.1 and SEQ ID NO.2. The present invention also provides a use of the aforementioned shRNAs in inhibiting the replication of the African swine fever viruses. The present invention also provides a recombinant expression vector containing the aforementioned shRNAs, and an adeno-associated virus (AAV) and a preparation method thereof. The provided shRNAs and recombinant AAV can be used to prepare drugs for preventing and / or treating African swine fever virus infection, such as therapeutic vaccines and can also effectively inhibit the replication and infection of the African swine fever viruses, and the preparation process is simple and conducive to large-scale production and promotion and application.

The invention discloses an African swine fever virus ELISA antibody detection kit, and belongs to the technical field of virus detection, the kit comprises an ELISA plate coated with antigens, and the antigens are prokaryotically expressed African swine fever virus p72, p30 and p54 proteins. The ELISA plate of the kit is coated with antigens, the antigens are prokaryotically expressed African swine fever virus p72, p30 and p54 proteins, the antigens are coated with extremely low antigen content, and the kit has no cross reaction with other main swine diseases and has good specificity through a specific test; the kit is high in sensitivity, good in repeatability and long in storage life, antibodies in different periods can be effectively detected through combined use of the three kinds of protein p72, p30 and p54 of the African swine fever viruses, a basis is provided for clinical swine herd infection conditions, and the kit plays an important role in preventing and controlling African swine fever virus infection.

The invention relates to an African swine fever virus p54 protein epitope peptide and application thereof. According to the invention, a new African swine fever virus p54 protein epitope is selected out through screening, the identification of the p54 protein N-terminal B cell epitope is completed, and the antigen epitope map of the p54 protein is improved. The selected antigen epitope (2DSEFFQPV9) is discovered for the first time, is highly conservative among various strains of African swine fever, and has important significance for serological detection of ASFV. The antigen epitope peptide and the antigen epitope peptide derived from the antigen epitope peptide and having the same function can be used for preparing a specific antibody of the African swine fever virus p54 protein, preparing a reagent for detecting or identifying the ASFVp54 protein, and laying a foundation for researching and developing vaccines for preventing and / or treating African swine fever virus infection.

The invention relates to the technical field of medicines, in particular to application of Triapine to treatment of African swine fever virus infection. The invention discloses application of Triapine and salt forms, hydrates, solvates and isotope substitutes thereof in preparation of drugs for treating African swine fever viruses. The invention has the following advantages: (1) Triapine has low toxicity to cells and is safe; and (2) Triapine has a good antiviral effect and can effectively treat African swine fever virus infection. (3) the selectivity coefficient is high, and safety is realized. And (4) the composition can be combined for use to enhance the treatment effect of African swine fever virus infection.

The invention discloses a method for rapidly detecting African swine fever virus by using a fluorescence in-situ detection technology. The method comprises the following steps: preparing a fluorescent probe, collecting and fixing cells of a sample to be detected, and hybridizing and dyeing the fluorescent probe and the cells, observing the dyed hybrid cells, and identifying whether the sample to be detected is infected by the African swine fever virus; signals are amplified through a primer pool method, a signal point is composed of thousands of DNA fragments, compared with a traditional in-situ hybridization method, the signals are stronger and easy to capture, a super-resolution imaging system is not needed, a signal cluster can be analyzed through a common fluorescence microscope, and the experiment cost and the operation difficulty are reduced.

The invention provides an African swine fever prevention and / or treatment neutralizing antibody which is an embedded monoclonal antibody and comprises a heavy chain and a light chain. A light chain variable region has a sequence shown as SEQ ID No:6, a heavy chain variable region has a sequence shown as SEQ ID No:2, and both a light chain constant region and a heavy chain constant region come froma swine derived antibody. The invention further provides a gene coding the embedded monoclonal antibody and an expression vector or a transformant comprising the coding gene. The expression carrier can be obtained by inserting the coding gene into pAAV-CAG. The invention further provides a method of utilizing the expression vector to prepare AAV. The antibody sequences or the transformant can beused for preparing detection reagents for African swine fever virus, agents for inducing immunoreaction aiming at African swine fever virus antigen in tested animals or agents for preventing animals from being infected by the African swine fever virus, and rapid and lasting antibody protection such as African swine fever virus vaccines can be provided.

The invention provides an African swine fever virus antibody ELISA (Enzyme Linked Immuno Sorbent Assay) detection kit. A support medium of the kit is coated with an African swine fever virus antigen,and the African swine fever virus antigen is African swine fever virus p30 and / or protein delta p54 protein. The African swine fever virus p30 protein is as shown in SEQ ID No. 1, and the African swine fever virus delta p54 protein is as shown in SEQ ID No. 2. The African swine fever virus antibody ELISA detection kit disclosed by the invention is good in specificity, sensitivity and repeatability, the sensitivity of the kit coated with the two antigens is equivalent to that of indirect immunofluorescence detection, antibody positive can be detected earlier at a low antibody level in the earlystage, a basis is provided for clinical swine herd infection conditions, and the kit plays an important role in preventing and controlling African swine fever virus infection.

The invention discloses application of a small molecule compound in anti-African swine fever virus ( ASFV) infection, and belongs to the technical field of medicine. According to the invention, an enzyme active center of E165R to exert hydrolytic action is defined by carrying out a crystal structure analysis of an E165R-dUMP complex, and then 1,2,3,4,6-O-Pentagalloylglucose capable of matching theE165R enzyme active center and significantly inhibiting E165R enzyme activity is obtained through in vitro screening by using the enzyme active center of E165R as a target. Further virus level experiments show that the 1,2,3,4,6-O-Pentagalloylglucose can significantly inhibit the replication of the ASFV in porcine alveolar macrophages, with high application value in clinical treatment or prevention of ASFV infection.

The invention belongs to the technical field of biology, and discloses an African swine fever virus CD2v truncated protein as well as application thereof in preparation of wild virus and natural attenuated virus detection kit Through comparison, it is found that positive serum infected by the African swine fever virus cannot be detected when prokaryotic expression CD2v truncated protein serves as the ELISA coating antigen; however, positive serum infected by the African swine fever virus can be detected when eukaryotic expression CD2v protein serves as the ELISA coating antigen. An indirect ELISA kit prepared by taking the eukaryotic expression CD2v protein as a coating antigen is matched with an indirect ELISA kit taking the African swine fever virus p54 protein as a coating antigen, so that the kit can be used for identifying infection of ASFV wild strains and natural attenuated strains; and moreover, the operation steps are simple, and the result is reliable. Therefore, the African swine fever virus indirect ELISA detection kit prepared from the eukaryotic expression truncated protein provided by the invention is very suitable for clinical large sample detection, and is suitable for large-scale popularization.

The invention relates to the technical field of medicines, in particular to application of Tilorone to prevention / treatment of African swine fever virus infection. The invention relates to application of telolone and salt forms, hydrates, solvates and isotope substitutes thereof in preparation of drugs for preventing or treating African swine fever viruses. The invention has the following advantages: (1) the bioavailability is high, and the half-life period is long; and (2) the maximum tolerance dose is high, the selectivity coefficient is high, and the safety is high. And (3) the activity of preventing / resisting African swine fever virus infection is strong. And (4) the drug delivery modes are multiple, and the application range is wide. And (5) the composition can be combined for use and has a good effect of preventing / treating African swine fever virus infection.