33 results about "Infectious hematopoietic necrosis virus" patented technology

The invention discloses a method for preparing a rainbow trout IHN (Infectious Haematopoietic Necrosis) inactivated vaccine, which comprises the following steps: carrying out grinding, filtering and poison dipping processing on pancreas and livers of juvenile fish which is attacked, but is still alive, inoculating rainbow trout gonad (RTG) cells, carrying out blind passaging at 14 DEG C, keeping for 5 days when carrying out blind passaging on the tenth generation, and collecting cell poisonous fluid; inoculating chinook salmon embryonic (CHSE) cells, carrying out passaging at 14 DEG C, and raising the culture temperature by 1 DEG C when passaging for 5 generations each time until the culture temperature is raised to 20 DEG C; and adopting epithelioma papulosum cyprini (EPC) cells to continuously carry out passaging under the condition of 20 DEG C, passaging to the twelfth generation to obtain high-titer virus solution and carrying out inactivation to obtain the rainbow trout IHN inactivated vaccine. According to the preparation method, RTG-2, CHSE-214 and EPC cells are utilized to alternately culture rainbow trout IHN viruses at a specific environment temperature so as to obtain a high-titer IHNV (Infectious Hematopoietic Necrosis Virus) antigen and produce the inactivated vaccine; and the technical difficult problem that the high-titer IHNV antigen cannot be obtained through single cells is solved.

The invention discloses a schisandra chinensis polysaccharide extract as well as a preparation method and application thereof. The preparation method of the schisandra chinensis polysaccharide extract sequentially comprises the following steps: (a1) taking schisandra chinensis, performing water extraction and collecting liquid phase, wherein ultrasonic pretreatment is conducted before water extraction; (a2) removing protein and micromolecular impurities and performing alcohol precipitation to obtain the schisandra chinensis extract. The invention also protects the schisandra chinensis extract prepared by the method. The invention also protects application of the schisandra chinensis extract to inhibition of IHNV (infectious hematopoietic necrosis virus). The invention provides certain theoretical basis for screening novel anti-IHNV medicines.

The invention discloses a cold-water-fish probiotics bacillus strain and application thereof. The strain HZC58, which has anti-virus, immune adjustment and immunity enhancement characteristics for cold water fishes, is separated and identified from an intestinal tract mucous membrane of a juvenile semi-wild rainbow trout in Benxi mountainous area of Liaoning province. According to colony morphological characteristic observation, 16SrDNA base sequence determination and homology analysis, the identification result of the strain indicates that the HZC58 belongs to bacillus and the collection number is CGMCC No.10706. In-vitro cell experiments prove that the bacillus strain HZC58 is cultured together with EPCs (endothelial progenitor cells) and has the characteristic of resisting IHNV (infectious hematopoietic necrosis virus) in EPC proliferation in vitro. Animal experiments prove that the bacillus strain HZC58 has the immune adjustment and immunity enhancement characteristics for the cold water fishes, and the strain HZC58 has also the anti-virus characteristic for the cold water fishes when being used for feeding individually. The bacillus strain HZC58 has a certain application prospect in cold-water-fish anti-virus, immunity adjustment and immunity enhancement.

The invention discloses an infectious hematopoietic necrosis vaccine and a method for amplifying infectious hematopoietic necrosis virus (IHNV) on epithelioma papulosum cyprini (EPC). The invention provides a method for preparing an infectious hematopoietic necrosis vaccine, and the method comprises the following steps: 1) inoculating IHNV to EPC according to an MOI value of 0.0001, carrying out culturing, and collecting supernatant, thereby obtaining proliferated IHNV; and 2) preparing the infectious hematopoietic necrosis vaccine by using the proliferated IHNV. Experiments in the invention prove that when the IHNV is inoculated to the EPC with the concentration of MOI = 0.0001, the required virus harvest time is short, and the virus titer is high and stable. The IHNV is amplified on a large scale based on the proliferation scheme, and an inactivated vaccine is prepared from BPL and formaldehyde. Results show that the vaccine has a good immune effect on Oncorhynchus mykiss, and the relative immune protection efficiency was up to 84%.

The invention discloses an infectious hematopoietic necrosis virus detection kit based on pyrosequencing. The infectious hematopoietic necrosis virus detection kit based on the pyrosequencing depends on an infectious hematopoietic necrosis virus fingerprint sequence, and the infectious hematopoietic necrosis virus fingerprint sequence is nucleotides at the 553th-565th positions of SEQ ID No.9 shown in a sequence table. The kit comprises a sequencing primer of the infectious hematopoietic necrosis virus fingerprint sequence and a primer pair for amplifying the infectious hematopoietic necrosis virus fingerprint sequence. The sequencing primer is single-stranded DNA represented by SEQ ID No.12 shown in the sequence table named as IHNV-S. The primer pair for amplifying the infectious hematopoietic necrosis virus fingerprint sequence consists of single-stranded DNA represented by SEQ ID No.10 shown in the sequence table named as IHNV-P and single-stranded DNA represented by SEQ ID No.11 shown in the sequence table.

The invention discloses a reagent for multiple detection of salmonidae virus and an application thereof. The reagent provided by the invention comprises a specific primer group, wherein the specific primer group comprises at least one pair from a first primer pair to a sixth primer pair; the first primer pair to the sixth primer pair are sequentially primer pairs of: nucleic acid of specifically-amplified spring viraemia of carp virus, nucleic acid of infectious hematopoietic necrosis virus, nucleic acid of viral haemorrhagic septicaemia virus, nucleic acid of infectious salmon anaemia virus,nucleic acid of salmonid alphavirus, and nucleic acid of infectious pancreatic necrosis virus. The upstream and downstream primers of the first primer pair to the sixth primer pair are sequentially the sequences as shown in Seq ID No.1 to Seq ID No.12. The reagent can carry out multiple detection on six salmonidae viruses, is simple and convenient to use, shortens the detection time of the six salmonidae viruses, improves the detection efficiency and quality, and has great significance for rapid detection and prevention and control of the six salmonidae viruses.

DNA constructs are provided which code for at least the extreme C-terminal amino acids of the rsaA protein of Caulobacter crescentus fused with heterologous polypeptides. Baterial cells containing, or which express the DNA constructs and secrete the resulting protein are also provided. Chimeric proteins including the C-terminal amino acids of the rsaA protein are provided, including chimeric proteins comprising antigenic epitopes of the Infectious Hematopoietic Necrosis Virus.

The invention provides a monoclonal antibody. The monoclonal antibody is prepared from salmons IHNV (infectious hematopoietic necrosis virus) immune animals, and has specificity on IHNV N protein. The invention also provides a salmons IHNV detection kit and a method, and application of the monoclonal antibody in preparing a product for diagnosing the salmons IHNV. The prepared IHNV monoclonal antibody has the advantages that the specificity is strong, and the infection of domestic J type salmons IHNV is detected, except U type; the crossing reaction with other rhabdovirus can be avoided; the detection fault and missing of the IHNV infected juvenile fishes by the existing molecular biology can be overcome; the results can be directly observed by a fluorescent microscope, the operation is simple, convenient and quick, the cost is low, the operation can be completed via the common laboratory, and the good application prospect is realized on the IHNV immune detection.

The invention discloses a high-efficient PCR (polymerase chain reaction) detection method of infectious hematopoietic necrosis virus and relates to a detection method of the infectious hematopoietic necrosis virus. The invention aims at the problem that the existing PCR method for inspecting the infectious hematopoietic necrosis virus is not high in sensitivity. The method comprises the following steps: 1) designing specific primers IHNV-Lf and IHNV-Lr; 2) getting a tissue filtrate after treatment of a detected pathological material; 3) preparing RNA (ribonucleic acid) of the IHNV (infectious hematopoietic necrosis virus); 4) performing PCR amplification to get an amplified product; and 5) observing the PCR amplified product and determining a result to end the detection. The detection method disclosed by the invention has great specificity, and the cross reaction with VHSV (viral hemorrhagic septicemia virus) does not exist; and according to the detection method disclosed by the invention, a polymerase protein is taken as a detection target, the specific primers IHNV-Lf and IHNV-Lr are utilized for detection, the operation is simple, the accuracy is high, the detection method is sensitive, the sensitivity under the condition of low concentration is great, the detection cost is relatively lower, and the detection method is more time-saving in comparison with an existing nested PCR (two-round PCR).

DNA constructs are provided which code for at least the extreme C-terminal amino acids of the <highlight><uline>rsa< / uline>< / highlight>A protein of Caulobacter crescentus fused with heterologous polypeptides. Baterial cells containing, or which express the DNA constructs and secrete the resulting protein are also provided. Chimeric proteins including the C-terminal amino acids of the <highlight><uline>rsa< / uline>< / highlight>A protein are provided, including chimeric proteins comprising antigenic epitopes of the Infectious Hematopoietic Necrosis Virus.

The invention discloses an anti-infectious hematopoietic necrosis virus (IHNV) monoclonal antibody 6G7, and preparation and application thereof. The monoclonal antibody 6G7 is secreted by a hybridoma cell strain IHNV-6G7; the preservation number of the cell strain is CCTCC C201360. The hybridoma cell strain IHNV-6G7 can stably generate the monoclonal antibody 6G7. The antibody has the advantages of being high in valence, high in sensitivity, strong in specificity, strong in affinity with natural antigen and the like. The invention also provides a quick test strip containing the monoclonal antibody, and a kit thereof. By adopting a fluorescent nanometer microspheres immune chromatography detection technology, the monoclonal antibody 6G7 has the characteristics of being good in sensitivity, specificity, stability, repeatability, reproducibility and the like. Meanwhile, the test strip and the kit are simple and convenient to use, high in test speeds, and applicable to a field test; the testing requirements of food safety, slaughter, testing mechanisms and the like can be met.

The invention discloses a biosensing detection method for identification of trout fries. The biosensing detection method is characterized in that on the basis of conservative analysis results of IHNV (infectious hematopoietic necrosis virus) gene sequence from different plant sources in a public database, six specific primers using IHNV virus matrix protein (M) encoding genes as amplification targets are designed, the constant-temperature reverse transcription amplification is performed on the to-be-measured total RNA template of the fish egg or fry sample, and the amplification time is only 15 to 30min; all or part of primers have the electrochemical activity markers in the amplification reaction, one part of dNTP has an anchor marker, the successfully amplified virus M gene sequence simultaneously has the electrochemical activity marker and the anchor marker, the amplified product can be captured onto the electrode surface by the anchor marker, and the carrying of IHNV in the sample can be accurately judged through detecting the electrochemical signal at the surface of the electrode. The biosensing detection method has the advantages that the operation is simple, the reaction is quick, the requirement on equipment precision is low, and the method is suitable for the site quick detection of production and business workplaces of fry farms, culture farms, and the like.

The invention discloses a high-efficient PCR (polymerase chain reaction) detection method of infectious hematopoietic necrosis virus and relates to a detection method of the infectious hematopoietic necrosis virus. The invention aims at the problem that the existing PCR method for inspecting the infectious hematopoietic necrosis virus is not high in sensitivity. The method comprises the following steps: 1) designing specific primers IHNV-Lf and IHNV-Lr; 2) getting a tissue filtrate after treatment of a detected pathological material; 3) preparing RNA (ribonucleic acid) of the IHNV (infectious hematopoietic necrosis virus); 4) performing PCR amplification to get an amplified product; and 5) observing the PCR amplified product and determining a result to end the detection. The detection method disclosed by the invention has great specificity, and the cross reaction with VHSV (viral hemorrhagic septicemia virus) does not exist; and according to the detection method disclosed by the invention, a polymerase protein is taken as a detection target, the specific primers IHNV-Lf and IHNV-Lr are utilized for detection, the operation is simple, the accuracy is high, the detection method is sensitive, the sensitivity under the condition of low concentration is great, the detection cost is relatively lower, and the detection method is more time-saving in comparison with an existing nested PCR (two-round PCR).

The invention discloses a method for preparing a rainbow trout IHN (Infectious Haematopoietic Necrosis) inactivated vaccine, which comprises the following steps: carrying out grinding, filtering and poison dipping processing on pancreas and livers of juvenile fish which is attacked, but is still alive, inoculating rainbow trout gonad (RTG) cells, carrying out blind passaging at 14 DEG C, keeping for 5 days when carrying out blind passaging on the tenth generation, and collecting cell poisonous fluid; inoculating chinook salmon embryonic (CHSE) cells, carrying out passaging at 14 DEG C, and raising the culture temperature by 1 DEG C when passaging for 5 generations each time until the culture temperature is raised to 20 DEG C; and adopting epithelioma papulosum cyprini (EPC) cells to continuously carry out passaging under the condition of 20 DEG C, passaging to the twelfth generation to obtain high-titer virus solution and carrying out inactivation to obtain the rainbow trout IHN inactivated vaccine. According to the preparation method, RTG-2, CHSE-214 and EPC cells are utilized to alternately culture rainbow trout IHN viruses at a specific environment temperature so as to obtain a high-titer IHNV (Infectious Hematopoietic Necrosis Virus) antigen and produce the inactivated vaccine; and the technical difficult problem that the high-titer IHNV antigen cannot be obtained through single cells is solved.

The invention discloses a schisandra chinensis polysaccharide extract as well as a preparation method and application thereof. The preparation method of the schisandra chinensis polysaccharide extract sequentially comprises the following steps: (a1) taking schisandra chinensis, performing water extraction and collecting liquid phase, wherein ultrasonic pretreatment is conducted before water extraction; (a2) removing protein and micromolecular impurities and performing alcohol precipitation to obtain the schisandra chinensis extract. The invention also protects the schisandra chinensis extract prepared by the method. The invention also protects application of the schisandra chinensis extract to inhibition of IHNV (infectious hematopoietic necrosis virus). The invention provides certain theoretical basis for screening novel anti-IHNV medicines.

The invention provides a method for increasing the artificial incubation success rate of rainbow trout fish eggs and the survival rate of fry. The method comprises the following step that in a fish egg incubation process, after the fish eggs enter the eyeing stage, povidone-iodine and formaldehyde are alternately added into water till incubation is finished. According to the method, povidone-iodine and formaldehyde are alternately added into the water from the eyeing stage of the fish eggs to the time when inoculation is completed so that the occurrence of water mildew can be prevented, infectious hematopoietic necrosis viruses and infectious pancreatic necrosis viruses on the surfaces of the fish eggs can be effectively killed, and accordingly the incubation success rate of the fish eggs and the survival rate of the fry are greatly increased.

The invention provides a salmon trout IHNV monoclonal antibody, which is obtained by secreting a monoclonal hybridoma cell line specific to IHNV N protein produced by immunizing animals with the infectious hematopoietic organ necrosis virus IHNV-BJLL strain. The invention also provides a detection kit and application based on the monoclonal antibody. Among the IHNV monoclonal antibodies obtained in the present invention, the monoclonal antibody secreted by a hybridoma cell line has strong specificity, can detect the infection of domestic U-type and J-type salmon trout IHNV at the same time, and can avoid the occurrence of other rhabdoviruses The cross-reaction overcomes the misdetection and missed detection of IHNV-infected juvenile fish by current molecular biology. The monoclonal antibody secreted by another strain is more specific and can detect U-type salmon trout IHNV infection, but it has no reaction with J-type salmon trout IHNV, so it can be used to identify the genotype of salmon trout IHNV in China. The detection method of the invention can directly observe the result through a fluorescence microscope, and this operation can be completed in a common laboratory, and has a good application prospect.

The invention discloses an application of IL-2 protein to preparation of an animal vaccine adjuvant. The amino acid sequence of the IL-2 protein is shown as sequence 2 in a sequence table. Experiments prove that compared with Oncorhynchus mykiss injected with IHNV (infectious hematopoietic necrosis virus) G (glycoprotein), the level of expression of IgT gene, Mx gene, Viperin gene, CD4 gene, CD8 gene and TNF-alpha gene of Oncorhynchus mykiss injected with the IHNV G and the IL-2 protein is increased substantially, the titer of a neutralizing antibody is increased remarkably, proliferation of lymphocytes is enhanced greatly, and the protective efficacy of the IHNV G is improved effectively. Therefore, the IL-2 protein has important application value in preparation of the animal vaccine adjuvant.

The invention discloses application of IL-17A protein in preparing animal vaccine adjuvants. The IL-17A protein is protein shown as an amino acid sequence 2 in a sequence table. Experiments prove that compared with rainbow trout injected with infectious hematopoietic necrosis virus G protein, expression level of IgT gene, IgM gene, Mx gene, Viperin gene and CD8 gene of the rainbow trout injected with the infectious hematopoietic necrosis virus G protein and the IL-17A protein is improved remarkably, valence of neutralizing antibody is improved remarkably, proliferation of lymphocytes is remarkably enhanced, and protection force of the infectious hematopoietic necrosis virus G protein is effectively increased. Therefore, the IL-17A protein has important application value in preparing the animal vaccine adjuvants.

The invention relates to PCR detection primers of prawn infectious hypodermal and hematopoietic necrosis virus. The primers have the nucleotide sequences: 5'-AATTTCTCCAAGCCTTCTCACC-3' of an upstream primer and 5'-TCTGGCAGCAAAGGTAACTCC-3' of a downstream primer. Compared with primers in the prior art, the PCR detection primers of the prawn infectious hypodermal and hematopoietic necrosis virus havethe advantage of a wider measurement range, during primer designing, an IHHNV sequence, obtained from a decapoda crustacean, published in GenBank is referred to, variation sites of IHHNV among different groups are avoided, the part which is conservative in published sequences separately is selected for primer designing, the measured range is wider and stabler, and a result is more accurate and reliable. The PCR detection primers of the prawn infectious hypodermal and hematopoietic necrosis virus can be applied for IHHNV pathogen detection and early warning in domestic prawn culture and seedling, and have good prospects.

The invention discloses application of IL-17A protein in preparing animal vaccine adjuvants. The IL-17A protein is protein shown as an amino acid sequence 2 in a sequence table. Experiments prove that compared with rainbow trout injected with infectious hematopoietic necrosis virus G protein, expression level of IgT gene, IgM gene, Mx gene, Viperin gene and CD8 gene of the rainbow trout injected with the infectious hematopoietic necrosis virus G protein and the IL-17A protein is improved remarkably, valence of neutralizing antibody is improved remarkably, proliferation of lymphocytes is remarkably enhanced, and protection force of the infectious hematopoietic necrosis virus G protein is effectively increased. Therefore, the IL-17A protein has important application value in preparing the animal vaccine adjuvants.

The invention discloses a reagent for multiple detection of salmonidae virus and an application thereof. The reagent provided by the invention comprises a specific primer group, wherein the specific primer group comprises at least one pair from a first primer pair to a sixth primer pair; the first primer pair to the sixth primer pair are sequentially primer pairs of: nucleic acid of specifically-amplified spring viraemia of carp virus, nucleic acid of infectious hematopoietic necrosis virus, nucleic acid of viral haemorrhagic septicaemia virus, nucleic acid of infectious salmon anaemia virus,nucleic acid of salmonid alphavirus, and nucleic acid of infectious pancreatic necrosis virus. The upstream and downstream primers of the first primer pair to the sixth primer pair are sequentially the sequences as shown in Seq ID No.1 to Seq ID No.12. The reagent can carry out multiple detection on six salmonidae viruses, is simple and convenient to use, shortens the detection time of the six salmonidae viruses, improves the detection efficiency and quality, and has great significance for rapid detection and prevention and control of the six salmonidae viruses.

The invention discloses a biosensing detection method for identification of trout fries. The biosensing detection method is characterized in that on the basis of conservative analysis results of IHNV (infectious hematopoietic necrosis virus) gene sequence from different plant sources in a public database, six specific primers using IHNV virus matrix protein (M) encoding genes as amplification targets are designed, the constant-temperature reverse transcription amplification is performed on the to-be-measured total RNA template of the fish egg or fry sample, and the amplification time is only 15 to 30min; all or part of primers have the electrochemical activity markers in the amplification reaction, one part of dNTP has an anchor marker, the successfully amplified virus M gene sequence simultaneously has the electrochemical activity marker and the anchor marker, the amplified product can be captured onto the electrode surface by the anchor marker, and the carrying of IHNV in the sample can be accurately judged through detecting the electrochemical signal at the surface of the electrode. The biosensing detection method has the advantages that the operation is simple, the reaction is quick, the requirement on equipment precision is low, and the method is suitable for the site quick detection of production and business workplaces of fry farms, culture farms, and the like.

The invention discloses a cold-water-fish probiotics bacillus strain and application thereof. The strain HZC58, which has anti-virus, immune adjustment and immunity enhancement characteristics for cold water fishes, is separated and identified from an intestinal tract mucous membrane of a juvenile semi-wild rainbow trout in Benxi mountainous area of Liaoning province. According to colony morphological characteristic observation, 16SrDNA base sequence determination and homology analysis, the identification result of the strain indicates that the HZC58 belongs to bacillus and the collection number is CGMCC No.10706. In-vitro cell experiments prove that the bacillus strain HZC58 is cultured together with EPCs (endothelial progenitor cells) and has the characteristic of resisting IHNV (infectious hematopoietic necrosis virus) in EPC proliferation in vitro. Animal experiments prove that the bacillus strain HZC58 has the immune adjustment and immunity enhancement characteristics for the cold water fishes, and the strain HZC58 has also the anti-virus characteristic for the cold water fishes when being used for feeding individually. The bacillus strain HZC58 has a certain application prospect in cold-water-fish anti-virus, immunity adjustment and immunity enhancement.

The invention discloses a primer and a kit for efficient triple detection of SVCV, IHNV and CEV. An improved LAMP technology is used as a gene amplification reaction principle, gold nanoparticles are added into a reaction system, ssDNA and protease are adsorbed, non-specific reaction in the heating process is inhibited, the purpose of hot start is achieved, and the non-specific reaction in the heating process is avoided. An improved LAMP technology and a micro-fluidic chip technology are combined, the carp spring viraemia virus, the infectious hematopoietic necrosis virus and the carp edema virus can be rapidly and accurately detected and distinguished, meanwhile, a reaction reagent is pre-embedded in the micro-fluidic chip, same three-index detection is achieved, and user operation is easy and convenient.

The invention belongs to the field of extracts, and particularly relates to preparation and application of an anti-IHNV kelp extract. The invention provides a method for preparing a kelp extract, which comprises the following steps: 1) preparing a kelp crude extract: removing protein in a kelp leaching solution to obtain the kelp crude extract; the kelp leach liquor is an extracting solution obtained by carrying out enzymolysis and water extraction on kelp, and 2) the kelp extract is obtained by carrying out ethanol precipitation on a dialysis retention solution and collecting precipitates, and the dialysis retention solution is in-bag liquid obtained by carrying out dialysis purification on the kelp crude extract by using a dialysis bag method. The invention further provides application of the kelp extract in preparation of an infectious hematopoietic necrosis virus inhibitor or an infectious hematopoietic necrosis virus resisting medicine. The inhibition rate of the kelp extract with the concentration of 300 mu g / mL to IHNV reaches 63.44%.

The invention discloses an infectious hematopoietic necrosis virus (INHV) RAA (recombinase-aid amplification)thermostaticfluorescence detection method and a detection kit. The detection kit includes aforward primer SEQ ID NO. 1,a reverse primer SEQ ID NO. 2,a specific fluorescent probe SEQ ID NO. 3,reaction liquid, reverse transcriptase, recombinant polymerase, and reference substance. The detection kit is high in specificity; detection sensitivity is high and can reach 0.1fg / [mu]L; high accuracyand reliability are realized; operation is easy, convenient, and quick, and the detection kit is suitable for field testing and has a wide range of application scenarios.

The invention discloses a fluorescent LAMP primer for detecting infectious hematopoietic necrosis virus of a fish, sequences of the fluorescent LAMP primer for detecting the infectious hematopoietic necrosis virus of the fish are shown as follows: SEQIDNO:1, SEQIDNO:2, SEQIDNO:3, SEQIDNO:4, SEQIDNO:5 and SEQIDNO:6. A fluorescent LAMP detection technology is established by introduction of a fluorescent dye based on an original LAMP technology, due to the use of instruments for fluorescence collection, the detection sensitivity is further improved, the fluorescent LAMP detection technology is more objective and more convenient to operate, and the fluorescent LAMP detection technology is easy to standardize and easy to popularize and use.

A method is provided for detecting the presence or absence of salmonid pathogens, including Infectious Hematopoietic Necrosis Virus (IHNV), Infectious Pancreatic Virus (IPNV), Infectious Salmon Anemia Virus (ISAV), Salmon Alphaviruses (SAV), Viral Hemorrhagic Septicemia Virus (VHSV), and Renibacterium salmoninarum. The method includes steps which may be carried out using a variety of analytical techniques, such as multiplexing RT-PCR, Target Specific Primer Extension (TSPE), and fluidic bead-based technology. PCR primers and TSPE primers which are components of the multiplex diagnostic assay using fluidic bead-based technology for detection of salmonid pathogens are also described.