81 results about "Necrovirus" patented technology

The invention discloses a fluorescent quantitative PCR detection kit for infectious spleen and kidney necrosis viruses and a fluorescent quantitative PCR detection method of the infectious spleen and kidney necrosis viruses. The detection kit and the detection method are characterized in that a specific primer and a probe are designed according to the sequence of a gene conservative area of the infectious spleen and kidney necrosis virus ORF007. The detection kit and the detection method have the advantages that the fluorescent quantitative PCR technology is adopted, the detection is simple, convenient and quick, the sensitivity is high, the specificity is strong, the repeatability is good, and the detection kit and the detection method can be applicable to monitoring of the infectious spleen and kidney necrosis viruses, and can also be used for detecting the virus content and the virus titer in siniperca chuatsi.

An RT-PCR detection method of a nervous necrosis virus relates to the field of virus detection. The invention provides an RT-PCR detection method of a nervous necrosis virus, an RT-PCR detection kit of nervous necrosis virus and a preparation method thereof. The RT-PCR detection method of the nervous necrosis virus comprises steps of: designing of an RT-PCR primer; construction of a recombinant plasmid pSP-NNV containing a detection target fragment, and preparation of a small RNA fragment as a positive control; an experiment of primer singularity and an experiment of sensitivity detection; and a recovery rate experiment, and a method validity experiment. The kit comprises a kit body and detection reagents, wherein the detection reagents comprise a lysate, an adsorption solution, a washing solution, redistilled water, an RT-PCR mixed reaction solution, an NNV- PCR reaction solution, a positive control, a negative control, an RNA extraction and an RT reaction tube. The preparation method of the kit comprises steps of: preparing the detection reagents; and putting the detection reagents in the kit body.

The invention provides a protein transduction domain derived from fish nervous necrosis virus. An amino acid sequence of the protein transduction domain is as shown in SEQ ID: 1. The protein transduction domain also provides a preparation method of the protein transduction domain, as well as use and application of the protein transduction domain to carrying allogenic materials to enter cells as well as preparing medicaments for controlling fish diseases. Compared with the prior art, the protein transduction domain provided by the invention has higher efficiency of entering fish cells.

The invention relates to an on-site rapid high-sensitivity detection kit of prawn infectivity muscle necrosis virus and a detection method thereof. The detection kit comprises a sampling tube, a rinsing tube filled with distilled water, a nucleate degeneration tube filled with a TE buffer solution, an amplification detection tube filled with amplification reaction liquid and nucleic acid dye, a negative control tube, a positive control tube, an FTA membrane, rapid drying liquid, and the like. Compared with the RT-PCR detection method, the detection method in the invention has the advantages of higher specificity, sensitivity and convenience, low cost, convenient use, more accurate and rapid detection and extremely sensitivity, is safe to both human and the environment and can substitute the traditional relevant detection method. The invention can be used in both the indoor laboratory and outdoor production field, has great significance for strengthening the epidemiological monitoring of prawn infectivity muscle necrosis virus, prawn cultivation waterbody monitoring and disease prevention and control and has great promotion and application value.

The present invention relates to genetic markers for discrimination and detection of viruses causing infectious aquatic organism diseases, and a method of discriminating and detecting the viruses using the same, and more particularly to a method for discriminating or detecting viruses causing infectious aquatic organism diseases, the method comprising: selecting and amplifying a DNA nucleotide sequence encoding a gene specific for viral hemorrhagic septicemia virus (VHSV), red sea bream iridovirus (RSIV) or infectious spleen and kidney necrosis virus (ISKNV), which is a virus causing red sea bream iridovirus disease, or Koi herpesvirus (KHV); hybridizing a peptide nucleic acid (PNA) that specifically recognizes the amplification product; controlling the temperature of the hybridization product to obtain a temperature-dependent melting curve; and discriminating the viral type or detecting whether or not fish would be infected with the viral type by analyzing the obtained melting curve to determine a melting temperature.

The invention discloses an ssDNA aptamer capable of specifically recognizing a GTONNV (Guangxi trachinotus ovatus nervous necrosis virus) and an application of the aptamer. The nucleotide sequence ofthe ssDNA aptamer is 5'-GTCTGAAGTAGACGCAGGAGCCTTTCGTGTTTCATTAGTGTGTTTCCATTGGGCGGCTCGGGGCAAAAGGAGTCACACCTGAGTAAGCGT-3'(SEQ ID NO: 1) or 5'-CCTTTCGTGTTTCATTAGTGTGTTTCCATTGGGCGGCTCGGGGCAAAAGG-3'(SEQ ID NO: 2). The ssDNA aptamer has specificity and high sensitivity to GTONNV infected cells and has no immunogenicity. The ssDNA aptamer has high specificity and high affinity, and is free of cytotoxicity,stable, easy to modify and convenient to synthesize and preserve, and the GTONNV infected cells can be detected and diagnosed rapidly and accurately.

The invention belongs to the technical field of virus detection, and specifically relates to a triple fluorescent PCR detection kit for the infectious spleen and kidney necrosis virus, largemouth bassranavirus and siniperca chuatsi rhabdovirus. The kit of the invention comprises virus-specific amplification primers and probes, a positive standard, a negative standard, Taq enzyme, reverse transcriptase, a RNA enzyme inhibitor, a reaction buffer, dNTP, nuclease-free water and a freeze-drying protective agent. The kit of the invention can perform multiple channel detection at the same time by using triple fluorescent PCR, uses the different fluorescently labeled probes, can detect the three viruses simultaneously in one reaction system, reduces the detection difficulty, shortens the detection time, and can help raisers accurately get detection results in time. The kit of the invention has high detection sensitivity, high stability and strong specificity, has no cross-reactions with otheraquatic viruses, and can be used for early monitoring and prevention and control of epidemic diseases.

The invention belongs to the technical field of biology, and relates to a method for preparing virus analogs of nervous necrosis viruses. The method comprises the following steps of: (1) performing expression of the virus analogs of the nervous necrosis viruses on escherichia coli of pQE30 plasmid vectors coded with nervous necrosis virus capsid protein genes under the pronucleus condition, wherein the expression is performed under the following conditions of: inoculating 1 mass percent of escherichia coli solution of which OD600 is equal to 1.5 into a culture medium, culturing the escherichia coli at 37 DEG C under 250rpm till the OD600 is 0.3 to 0.5, adding isopropyl-beta-D-thiogalactoside into the escherichia coli solution till the final concentration of the isopropyl-beta-D-thiogalactoside is 900muM, transferring the solution, and continuously culturing the escherichia coli for 3 to 4 hours at the temperature of between 25 and 30 DEG C at the rotational speed of 200rpm to finish the expression; and (2) after the expression is finished, breaking, separating and purifying the strains to obtain the virus analogs of the nervous necrosis viruses, wherein the plasmid coded genes also can be nervous necrosis virus capsid protein genes containing histidine tags, and the expression product of the genes can be purified by affinity chromatography. The method provided by the invention can obtain high virus analog expression amount; and the chromatography and purification method is simple and convenient and has low costs in required apparatuses and reagents.

The invention discloses a proliferation method of a siniperca chuatsi infectious spleen and kidney necrosis virus (ISKNV). According to the proliferation method, synchronous virus inoculation is adopted, the virus and a siniperca chuatsi brain tissue cell line (CPB) are in a suspended state, and the mutual contact surface area between the virus and the siniperca chuatsi brain tissue cell line is large so that the virus enters cells through more space receptors; and the virus infects the cells while the cells differentiate and proliferate, thus obtaining the high content ISKNV. After the CPB is cultured for 30 hours to 60 hours, the CPB is transferred to 5%-8% v / v of fetal calf serum culture medium, and the ISKNV is synchronously inoculated at the MOI (Multiplicity of Infection) value of 0.85-2.3, thus obtaining a low-cost ISKNV proliferation method. After the CPB is cultured for 20 hours to 40 hours, the CPB is transferred to 8%-10% v / v of fetal calf serum culture medium, and the ISKNV is synchronously inoculated at the MOI (Multiplicity of Infection) value of 30-45, thus obtaining a short ISKNV proliferation method. Cultivation methods can be selected according to existing conditions. The proliferation method provides theoretical basis for production of low-cost viruses.

The invention discloses RT-LAMP detection primer pairs, a detection kit and a detection method for infectious haematopoietic necrosis virus (IHNV). The detection primer pairs comprises a pair of outer primers, a pair of inner primers and a pair of loop primers; the detection kit comprises primer liquid, reaction liquid, DNA polymerase, reverse transcriptase and control; and the detection kit also can contain a color developing agent. The detection method comprises the following steps: extracting to-be-detected virus RNA, and amplifying a sample RNA template at the temperature of 63-65 DEG C by utilizing reverse transcription activity of reverse transcriptase and adopting six specific primers and one DNA polymerase with strain displacement activity, wherein the pg grade of pure virus RNA can be detected; and identifying by adding SYBR Green I ESE-Quant-tube Scanner instrument detection, or utilizing a turbidity meter for observing change of turbidity of sediments in a reaction tube, so as to judge whether to carry out amplification or not and determine whether the to-be-detected sample contains IHNV RNA or not. The RT-LAMP detection primer pairs, the detection kit and the detection method for the IHNV have the advantages of quickness, high efficiency, easy operation, high specificity, high sensitivity, easy identification and applicability to field detection and are applicable to popularization and application.

The invention provides a simple, fast, high sensitivity and easy operation method for detection of fish nervous necrosis viruses, and a portable on-site usage kit composed of detection reagents and devices. The primer set used by the method has nucleotide sequences of SEQ ID NO: 1-5. The method and kit are used for red-spotted grouper nervous necrosis virus detection for non-disease diagnosis and treatment, have the advantages of operation easiness, short time consumption and high sensitivity, are suitable for on-site detection and monitoring of fish nervous necrosis viruses in a culture enterprise and various-grade aquatic animal disease prevention and control parts, and have a high practical value and a good application prospect.

The invention relates to a kit for rapidly detecting an infectious pancreatic necrosis virus of rainbow trout by utilizing a loop-mediated isothermal amplification (LAMP) technique and a detection method of the infectious pancreatic necrosis virus of rainbow trout. The kit comprises reaction liquid A and reaction liquid B, wherein the reaction liquid A contains 10*isothermal reaction buffer liquid, 5U / microliter of AMV, 20U / microliter of Rnasin, 8U / microliter of Bst DNA (Deoxyribose Nucleic Acid) polymerase, 10mM of dNTP (deoxy-ribonucleoside triphosphate), 25mM of magnesium sulfate, 20 micrometers of inner primers 1, 20 micrometers of inner primers 2, 10 micrometers of outer primers 1, 10 micrometers of outer primers 2, 30 micrometers of circular primers 2 and 5M of lycine; the reaction liquid B is 1000*fluorochrome SYBR (Synergy Brands) GreenI. According to the kit, the rapid detection of the infectious pancreatic necrosis virus is realized by carrying out methods of the RNA (Ribose Nucleic Acid) extraction in tissue samples or cell culture liquid, the loop-mediated isothermal amplification of the infectious pancreatic necrosis virus, the color development and detection of amplified products and the like. With the adoption of the technique, the defects that the detection time is long, the operation is complicated, expensive instruments are required, and the like in the existing technical detection are overcome, and the technique is suitable for rapid spot detection in livestock farms, veterinary stations and the like.

The invention provides a recombinant Lactobacillus casei for expressing the infectious pancreas necrosis virus (IPNV) VP2 protein and a preparation method thereof. According to the whole gene sequence of the IPNV VP2 protein and the gene fusion characteristic of expression vector plasmid, a pair of primers are designed to perform PCR to obtain target segment of 1095bp containing the main antigen site of the IPNV VP2 protein, the IPNV VP2 protein obtained through amplification is linked to the surface expressed carrier plasmid pPG1 and enters the cell of the host strain Lactobacillus casei 393 through electrotransformation, and the Lactobacillus casei containing positive recombinant plasmid pPG1-IPNV VP2 is named pPG1-IPNV VP2 / L. casei 393. The recombinant pPG1-IPNV VP2 / L. casei 393 is expressed under the induction of lactose. In the invention, the Lactobacillus casei expression carrier system of the IPNV VP2 protein is constructed and the target protein of around 40 KDa containing the main antigen site of the IPNV VP2 protein is expressed, and according to the Western blot experiment and indirect immunofluorescence experiment, the expressed foreign protein is capable of reacting with the IPVN immune serum, which shows that the recombinant VP2 protein and the IPNV natural antigen have the same antigenicity.

Existing commercial fish nervous necrosis virus vaccine must be vaccinated by injection. The invention relates to a gene recombinant fusion protein MCP-ompN1 of a major coat protein (MCP) of nervous necrosis virus and ompN1, having a transmembrane effect, of edwarsiella ictaluri as well as expression and a preparation method of the fusion protein. The MCP and ompN1 protein are flexibly linked by asequence composed of four amino acids ''GGGS''". A nucleotide sequence encoding MCP-ompN1 is first optimized according to the codon preference of a BL21-DE3 engineering strain, then an expression plasmid is constructed and expressed in the BL21-DE3 strain, and then the MCP-ompN1 fusion protein is separated and purified through bacterial disruption, inclusion body treatment, protein renaturation,nickel column purification, removal of histone tags with biotin-labeled thrombin, removal of thrombin with avidin resin and other steps. The gene recombinant MCP-ompN1 fusion protein can be used as avaccine antigen of nervous necrosis virus, or a vaccine antigen of nervous necrosis virus and edwarsiella ictalurid, and the ompN1 protein in the MCP-ompN1 fusion protein mediates mucosal immunity.

The invention discloses a method for selecting nervous necrosis virus resistant individuals of epinephelus lanceolatus. The method comprises the following steps: (1) obtaining SNP site information of the epinephelus lanceolatus; (2) analyzing the disease resistance weight value of the SNP marker and selecting the SNP marker; (3) obtaining candidate epinephelus lanceolatus individual SNP site information and selecting individuals with nervous necrosis virus resistance; and (4) detecting the selection accuracy of disease-resistant individuals. According to the method, the SNP marker with the maximum nervous necrosis virus resisting weight is screened from the SNP markers of the epinephelus lanceolatus, so that a method capable of selecting nervous necrosis virus resisting individuals is established.

The invention discloses an ssDNA nucleic acid aptamer specifically targeting Trachinotus ovatus nerve necrosis virus and application thereof, wherein the nucleotide sequence of the ssDNA nucleic acidaptamer is 5'-GTCTGAAGTAGACGCAGGAGAACTGTATTAGCCTCTGGGTGCCGCACCGTAGTGCCCTATCTAACATCACAGTCACACCTGAGTAAGCGT-3' (SEQ ID NO: 1) or 5'-AACTGTATTAGCCTCTGGGTGCCGCACCGTAGTGCCCTATCTAACATCAC-3' (SEQ ID NO:2). The ssDNA nucleic acid aptamer of the invention has specificity and high sensitivity to nerve necrosis virus infected cells of Trachinotus ovatus , and has no immunogenicity. The ssDNA nucleic acid aptamer has high specificity, high affinity, no cytotoxicity, stable and easy to modify, easy to synthesize and preserve, and can be used for rapid and accurate detection and diagnosis of nerve necrosis virus infected cells of Trachinotus ovatus.

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 a preparation method of a rainbow trout infectious haematopoietic necrosis virus (IHNV) inactivated vaccine. The method comprises the following steps of S1, recovering and purifying an FHM cell; S2, basically culturing the FHM cell; S3, amplifying and culturing the FHM cell; S4, culturing an IHNV virus; S5, purifying the IHNV virus; S6, inactivating the IHNV virus. The IHNVinactivated vaccine prepared through the method is applied to preventing infectious haematopotietic necrosis; the IHNV inactivated vaccine successfully prepared through the method is good in inactivation effect, high in safety and high in relative immune protective rate; meanwhile, the preparation method is simple in process, beneficial to reducing a large-scale production cycle and cost of the virus, and capable of being used for industrially preparing the IHNV inactivated vaccine for preventing infectious haematopotietic necrosis.

The invention discloses an infectious clone of grape berry necrosis virus and a construction method thereof. The infectious cloning vector is obtained by connecting segmented amplified grape berry necrosis virus (GBNV) genomic fragment to pCB301-2x35S-HDVRZ-NOS carrier containing double 35S promoter and ribozyme HDV-RZ in a seamlessly clone. The vector was transfected into competent cells of Agrobacterium tumefaciens EHA105 by freeze-thaw method, and its infectivity was tested by inoculating western tobacco and grape. The GINV infectious clone constructed by the invention can be stably and efficiently expressed in Agrobacterium tumefaciens EHA105 strain, and can successfully infect western tobacco and grape hosts. The cloning of GINV infectious clones makes the study of GINV easy to operate at DNA level, and provides a powerful tool for the further study of biological characteristics and pathogenicity of GINV.

The invention discloses a specific primer pair for testing an infectious splenorenal necrosis virus. The invention also discloses a probe used in combination with the primer pair. The invention also discloses a test kit. The invention takes the DNApolymerase gene in an infectious splenorenal necrosis virus as a test target, uses the isothermal amplification technique, and adopts a combination of aspecific primer and a probe, which improves the test convenience and specificity of the infectious splenorenal necrosis virus, and also greatly shortens the test time. Compared with the PCR test method, the method disclosed by the invention saves the product electrophoresis verification process, and avoids the false positive result, which improves the test accuracy. Compared with qPCR, the methoddisclosed by the invention is simple and easy to operate, and does not need to operate complicated instruments and equipment, which saves costs, improves the test efficiency, and is convenient to popularize and use in a wide range. Compared with other isothermal amplification methods, the test method disclosed by the invention requires less time and has a higher test accuracy.

The invention discloses a primer group for detecting nervous necrosis viruses of fishes and application thereof, belongs to the technical field of biological detection and provides a primer group designed according to highly-conservative regions of Coat protein gene sequence of the nervous necrosis viruses of fishes. The primer group has the characteristics of high specificity and high sensitivity; the primer group is prepared into a kit for detecting nervous necrosis viruses of fishes based on the characteristics of the primer group; a loop-mediated isotherm amplification (LAMP) method is used for detecting nervous necrosis viruses of fishes, so the method has the characteristics of simplicity in operation, rapid reaction and visible reaction results.

The invention discloses a fluorescent quantitative PCR method for detecting infectious spleen and kidney necrosis virus of mandarin fish and a corresponding kit. Specific gene detection is ingeniouslyapplied to distinguish the infectious spleen and kidney necrosis virus of the mandarin fish from strains or viruses of other species, and accurate bacterial genus information is obtained through comprehensive determination. Compared with an existing mainstream detection kit, the kit for detecting the infectious spleen and kidney necrosis virus of the mandarin fish has the advantages of being highin sensitivity, rapid, convenient to use, good in specificity, rigorous and accurate in judgment and the like, and has good application prospects and market value.

The invention discloses an antibody for detecting infectious spleen and kidney necrosis viruses as well as preparation and application thereof. The antibody for detecting infectious spleen and kidney necrosis viruses is a purified anti-rabbit ISKNV-23P8 polyclonal antibody which is capable of specifically recognizing ISKNV-23P8, obtained by adopting an amino acid residue containing a sequence having a sequence number of SEQ ID NO: 1 as an epitope. The antibody is used for detecting the infectious spleen and kidney necrosis viruses and has specificity. An immunofluorescence detection kit for the infectious spleen and kidney necrosis viruses and a non-diagnostic immunofluorescence rapid detection method for infectious spleen and kidney necrosis virus-infected imprints have the characteristics of good specificity, simple operation, quickness and sensitivity, have obvious advantages compared with a PCR method, and can be used for ISKNV infection detection during the culture of various susceptible fishes; and all operations can be completed within 1.5 hours.

The present invention is related to a low cost, fast, specific, sensitive, and suitable for routine application in monitoring activities, procedure for determining variants of infectious pancreatic necrosis virus (IPNV) on samples of different origin; and associated kit.

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 construction method and application of a vector vaccine for resisting infectious spleen and kidney necrosis viruses, and belongs to the technical field of virus genetic engineering. The vector vaccine comprises an antigen protein MCP expressed by a bombyx mori nuclear polyhedrosis virus and shown as SEQ ID NO: 10, and the antigen protein is encoded by a cmv-mcp expression cassette shown as SEQ ID NO: 1. According to the construction method, the mcp expression cassette cmv-mcp controlled by a cytomegalovirus promoter cmv is cloned to a transfer plasmid to construct a recombinant plasmid, then competent cells are transformed to obtain Bacmid-MCP, the Bacmid-MCP DNA transfects bombyx mori culture cells to obtain BmNPV-MCP, then the BmNPV-MCP is inoculated to larvae or primary pupae of bombyx mori at the age of 4-5, and homogenate is carried out after the disease is attacked to obtain the vector vaccine. The vaccine is used for immunizing siniperca chuatsi / weever through an injection, oral administration or soaking method, and the occurrence of infectious spleen and kidney necrosis diseases can be reduced.

The invention discloses an infectious spleen and kidney necrosis virus (ISKNV) gene intron and its use in distinguishing of live and inactivated ISKNVs and belongs to the field of virus detection. The intron is located on ORF003L of an ISKNV genome NC_003494.1, has length of 80bp and is named as IN-3. The invention discloses the intron of ISKNV. The intron is named as IN-3. Through the characteristics of transcription shearing of the intron IN-3, the intron IN-3 is used as a virus complete inactivation index, and an ISKNV inactivation rapid detection nested RT-PCR method is established. The ISKNV gene intron can shorten an inactivation test cycle in ISKNV cell inactivated vaccine production process and improve vaccine production efficiency.

The invention discloses a method for detecting an infectious haematopoietic necrosis virus based on liquid-phase chip. The invention provides a specific primer pair for identifying the infectious haematopoietic necrosis virus in an auxiliary manner, wherein the specific primer pair is composed of a single-chain DNA (Deoxyribonucleic Acid) molecule as shown in sequence 1 of a sequence table and the single-chain DNA molecule as shown in sequence 2 of the sequence table. The invention further protects a primer probe composition for identifying the infectious haematopoietic necrosis virus in the auxiliary manner, wherein the primer probe composition is composed of the specific primer pair and a probe T1; and a nucleotide sequence of the probe T1 is as shown in sequence 3 of the sequence table. The specific primer pair provided by the invention has good specificity in identifying the infectious haematopoietic necrosis virus. The primer probe composition provided by the invention is combined with the liquid-phase chip to identify the infectious haematopoietic necrosis virus, and therefore, the method has the advantages of being good in specificity, high in sensitivity, simple to operate, short in needed time, free of environmental pollution, free of health risks to the people and capable of carrying out high-flux detection.