203 results about "Duck hepatitis A virus" patented technology

The invention relates to an enzyme-linked immunosorbent assay (ELISA) kit for duck hepatitis virus type-I serum antibody and relates to a test method and application of the kit. The kit comprises an enzyme label plate coated by the recombinant VP1 (virus protein) protein, a rabbit anti-duck IgY antibody marked by horseradish peroxidase, a TMB substrate colour reagent, a positive serum, a negative serum and a kit specification. In the invention, by adopting the polymerase chain reaction, the VP1 genes are amplified from the DHV-1genome and the VP1 gene-containing recombinant expression plasmid pET32a-VP1 is constructed; the plasmid is transferred to host cells BL21 (DE3), and the in-vitro expression VP1 protein is purified by a nickel column and then used as the antigen; the enzyme-linked immunosorbent assay kit is established; the positive serum is the standard positive serum of duck hepatitis virus type-I and the negative control is the standard negative serum of duck. The test kit has the advantages of strong specificity, high sensitivity, simple operation, easy large-scale popularization and application, very important application value in diagnosis of duck hepatitis virus type-I, survey of epidemiology and immunization survey and the like.

The invention provides a dual fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) specific primer for detecting type 1 and type 3 duck hepatitis A viruses. A dual fluorescence quantitative method comprises the following steps: comparing sequences of DHAV-1 and DHAV-3 published on GenBank, and designing a pair of specific detection primers SEQ1 and SEQ2 aiming at DHAV-1 and a Taqman probe (probe1) as well as a pair of specific detection primers SEQ3 and SEQ4 aiming at DHAV-3 and a Taqman probe (probe2) respectively in a region with conservation and relatively large difference between gene sequences of the two viruses; confirming the concentration of the dual fluorescence quantitative RT-PCR specific primer and the Taqman probe aiming at DHAV-1 and DHAV-3. The dual fluorescence quantitative method built by virtue of the group of primers is good in specificity and high in sensitivity, and can be used for quick serum type identification and real-time quantitative analysis of the type 1 and type 3 duck hepatitis A viruses.

The invention discloses a Korean novel duck hepatitis viral antibody ELISA (Enzyme-Linked Immunosorbent Assay) detection kit. The detection kit contains an ELISA board coated by Korean novel duck hepatitis VP1 (Phenotypic Variance1) recombination protein, a sample diluent, concentrated washing liquid, an enzyme conjugate working solution, a chromogenic reagent (A), a chromogenic reagent (B), a stopping solution, a positive contrast solution and a negative contrast solution. The VP1 recombination protein is obtained by using the following method: using Korean novel duck hepatitis viruses as a material, augmenting and cloning the VP1 gene through an RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction) method to obtain recombinant expression plasmid pMD (physical medium dependent)-VP1; then, directionally inserting to an expression vector pET-32a (+) and screening to obtain recombinant expression plasmid pET-32a(+)-VP1; and inducing, expressing and purifying by ITPG (Isopropyl beta-D-Thiogalactopyranoside) to obtain VP1 recombination protein. The detection kit is used for detecting the Korean novel duck hepatitis and has strong specificity, high sensitivity, simplicity of operation, easiness of popularization and application in a large-area range and wide market prospects.

The invention discloses a one-step dual RT-PCR detection kit of 1-type and 3-type duck hepatitis A virus, primer pairs and a method thereof. The primer 1 is composed of a primer 1 and a primer 2 which are used in detection of 1-type duck hepatitis A virus, and nucleotide sequences of the primer 1 and the primer 2 are respectively single chain DNA as shown in SEQ ID NO:1 and SEQ ID NO:2. The primer pair 2 is composed of a primer 3 and a primer 4 which are used in detection of 3-type duck hepatitis A virus, and nucleotide sequences of the primer 3 and the primer 4 are respectively single chain DNA as shown in SEQ ID NO:3 and SEQ ID NO:4. The detection technology for simultaneously detecting two pathogens of 1-type and 3-type duck hepatitis A virus by one RT-PCT reaction is adopted to avoid repeated conventional PCR detection, and has advantages of low cost, high efficiency and the like. In addition, amplification results are directly determined by the utilization of different lengths of amplified fragments for the design of the primers such that the method is simpler, more visual and practical during result determination.

The invention discloses a duck egg yolk antibody for preventing and treating virus hepatitis of ducklings, and a preparation method thereof. The duck egg yolk antibody is extracted from the primiparous eggs of a high-immunity homebred breed duck starting laying for one month. The preparation method comprises the following steps of: (a) highly immunizing the breed duck before laying; (b) collecting high-immunity primiparous breed duck eggs; (c) cleaning and sterilizing; (d) separating egg yolks; (e) diluting; (f) stirring and filtering; and (g) packaging and preserving. After the egg yolks are continuously diluted for 2 times by using normal saline, the DHV (Duck Hepatitis Virus) precipitating antibody is detected by a conventional method, the antibody titer is more than 1:64, and the HI titer of H5 is more than 1:512. 0.5mL of the duck egg yolk antibody is subcutaneously injected to a 3-day-old duckling for preventing virus hepatitis; and 1mL of egg yolk antibody to which antibiotics are added is subcutaneously injected to a duck for treating virus hepatitis, and the egg yolk antibody is re-injected to a seriously diseased duck every other day.

The invention discloses a DHAV (duck hepatitis A virus) typing detection method based on a fluorescent quantitative PCR (polymerase chain reaction) melting curve method. The DHAV typing detection method comprises steps as follows: virus RNA (ribonucleic acid) extraction and cDNA (complementary deoxyribonucleic acid) synthesis; preparation of positive standard forms; establishment of the DHAV typing detection method based on the fluorescent quantitative PCR melting curve method; method validation adopting a specificity test, a sensitivity test and a reproducibility test. According to the DHAV typing detection method, all that is required is to add a pair of primers, A and C gene types are detected by single tube, the method is simple and feasible, and time consumption is low; the whole amplification and detection process adopts closed tube operation, so that pollution of PCR amplification products is avoided, and false positive results are reduced; amplified fragments are short, higher annealing temperature is adopted, and amplification specificity is guaranteed. The fast DHAV typing detection method is established, great convenience is provided for correct diagnosis and control of the DHAV, and economic benefits of agricultural production are achieved.

The invention discloses a molecular biological identification method for I type duck hepatitis virus, which comprises the steps of treating samples to be tested, preparing RNA of the samples, obtaining cDNA of the samples by reverse transcription, identifying I type duck hepatitis virus by the fluorescent quantitive RT-PCR method or the nested PCR method, etc. The molecular biological identification method has the advantages of fast diagnosis, high sensitivity and wide applied range, can be used for identifying DHV-1 from the tissue samples, cotton swabs, ducks or chicken embryo allantoic liquid or cell cultures.

The invention discloses a duck hepatitis virus type I LAMP (loop-mediated isothermal amplification) detection kit. A LAMP technology is adopted; and two specific inner primers, two specific outer primers and two specific ring primers are designed according to a gene conserved region shared by duck hepatitis viruses type I. Due to the application of the duck hepatitis virus type I LAMP detection kit and a detection method established by the invention, the defects of long detection time, large workload, cross pollution, complex operation and the like in the prior art are overcome. The duck hepatitis virus type I LAMP detection kit has the strong specificity and high sensitivity, is rapid, has low cost and simpler operation method and is particularly suitable for the requirement on field rapid detection of the duck hepatitis virus type I in the primary-level areas.

The invention relates to duck virus hepatitis inactivated vaccine and a preparation method thereof. The preparation method comprises the following steps: taking a duck hepatitis virus virulent strain CGMCC No.3202 with excellent immunogenicity and obtained by the inventor through field separation as a viral strain for producing vaccine; inoculating the viral strain in a susceptible chick embryo and obtaining the embryo fluid; and carrying out emulsification after inactivation to prepare the safe effective duck virus hepatitis vaccine. The duck virus hepatitis inactivated vaccine aims to prevent duck virus hepatitis virus infection and fills a gap in the market, thereby providing conditions for controlling the spread of duck virus hepatitis on poultry farms.

The invention discloses a novel DHAV (duck hepatitis A virus) JS strain and application of the DHAV JS strain in duck virus hepatitis prevention and cure. The DHAV JS strain is obtained through the steps of separation culture, duck embryo neutralization test, PCR (polymerase chain reaction) detection of viruses and sequencing detection experiment of the viruses, and the strain is determined to be the DHAV serotype 3; and the microbial preservation number is CGMCCNo.6852. According to the invention, through taking the novel DHAV JS strain as the virus strain for producing vaccine, the novel DHAV inactivated vaccine is prepared through the steps of inoculating 10-day-old SPF (specific pathogen free) duck embryos, selecting the embryos died after incubating for 48 hours, collecting the embryo liquid, inactivating and emulsifying. The clinical application proves that the prepared inactivated vaccine is favorable in safety and conducive to prevention and control of the novel DHAV disease, can prevent the infection and outbreak of the novel DHAV in a targeted way, and can be in mass production and clinical application.

The invention discloses a multiple PCR (Polymerase Chain Reaction) detection kit for duck hepatitis A virus 1 and 3 as well as MDPV (Muscovy Duck Parvovirus). The detection kit contains three pairs of specific primers. Experimental results show the multiple PCR detection kit for duck hepatitis A virus 1 and 3 as well as MDPV can be applied to detecting the duck hepatitis A virus 1 and 3 and the MDPV. The multiple PCR detection kit has the advantages of being convenient to operate, high in sensitivity, strong in specificity and the like; furthermore, the multiple PCR detection kit can be clinically used for distinguishing and detecting the duck hepatitis A virus 1 and 3 and the MDPV, the time cost can be saved, and the pollution can be reduced.

The invention discloses a multi-fluorogenic quantitative PCR (polymerase chain reaction) method for detecting a duck hepatitis A virus (DHAV) and identifying DHAV-A and DHAV-C and a kit, and belongs to the technical field of biological detection. Aiming at a DHAV3D area, the invention designs a pair of conservative amplification primers (SEQIDNO:1 and SEQIDNO:2) and a conservative probe (SEQIDNO:5) to detect different gene types of DHAVs. A pair of specific primers (SEQIDNO:3 and SEQIDNO:4) and two specific probes (SEQIDNO:6 and SEQIDNO:7) designed by aiming at DHAV5'UTR are used to identify DHAV-A and DHAV-C. The method has excellent specificity, repeatability and sensitivity, is convenient, simple and quick in operations, is used for identifying and diagnosing the infection of DHAV as well as DHAV-A and DHAV-C in scientific research and clinical application, and is also used for the epidemiological investigation of DHAV.

The invention belongs to the technical field of molecular biology and particularly relates to NDV (Newcastle disease virus) recombinant virus expressing DHAV-1 and DHAV-3 VP1 genes.The recombinant virus is recorded as rLS-1VP1-2A-3VP1 and is obtained inserting serially connected DHAV-1 and DHAV-3 VP1 genes into an NDV vector and carrying out saving.The NDV (Lasota strain) is used as the vector for the recombinant virus, the serially connected DHAV-1 and DHAV-3 VP1 genes are inserted into the NDV (Lasota strain) to obtain a vector, and the DNV recombinant virus co-expressing DHAV-1 and DHAV-3 VP1 genes is obtained by determining an optimal insertion site to insert the DHAV VIP1 gene; the recombinant virus is useful in preventing duck hepatitis A viruses (type 1 and type 3) and duck Newcastle disease and filling the current blank of DHAV-3 vaccines.

The invention discloses a general type duck hepatitis A virus antibody ELISA (Enzyme-Linked Immunosorbent Assay) detection kit which comprises an ELISA plate coated with DHAV (Duck Hepatitis A Virus)-1VP3-3VP1 recombinant protein. The DHAV-1VP3-3VP1 recombinant protein is obtained through a method which comprises the following steps: respectively amplifying and cloning a DHAV-1VP3 gene and a DHAV-1VP1 gene by taking DHAV-1 and DHAV-3 as materials to obtain recombinant plasmids pMD-1VP3 and pMD-3VP1; then directionally inserting into a pET-32a(+) expression vector in series, and screening to obtain a recombinant expression plasmid pET-1VP3-3VP1; and carrying out ITPG (Isopropyl beta-D-thiogalactoside) inducible expression and purification to obtain the DHAV-1VP3-3VP1 recombinant protein. The general type duck hepatitis A virus antibody ELISA detection kit disclosed by the invention can be used for evaluating the levels of DHAV-1 and DHAV-3 antibodies through primary detection and is low in cost, easy, convenient and fast to operate and suitable for the detection of batch samples.

The invention discloses an indirect ELISA method and kit for detecting serum 3-type duck hepatitis virus a antibody and belongs to the technical field of serum antibody detection. The indirect ELISA method includes that serum 3-type duck hepatitis virus VP1 protein is utilized as an envelope antigen with the peridium quantity as 0.1-1mug per hole, HRP-mice anti-duck IgY is utilized as the HRP with the use concentration as 0.2-2mug/mL, and the coloration time is 10 minutes. The kit comprises a VP1 protein antigen envelope board, the HRP-mice anti-duck IgY, sample diluent, a scrubbing solution, TMB solutions A and B and a stop solution. The method and the kit can be used for detecting the serum 3-type duck hepatitis virus a antibody in duck serum and duck egg yolk, and the serum does not have cross reaction with positive serum of other viruses. Compared with a traditional neutral reaction detection method, the method has the advantage of being convenient and accurate.

The invention discloses a reagent kit for identifying duck hepatitis A virus infection and novel duck reovirus infection. Specific primers (such as SEQ No. (sequence number) 1-4) of a duck hepatitis A virus and a novel duck reovirus are adopted to establish a double PCR (Polymerase Chain Reaction) detection method, and assembled into the reagent kit. The reagent kit can identify the duck hepatitis A virus and the novel duck reovirus in the same system and the mixed infection of the duck hepatitis A virus and the novel duck reovirus. The reagent kit has the characteristics of quickness, accuracy, high sensitivity, good specificity and the like, and can well solve the problem that the duck hepatitis A virus infection and the novel duck reovirus infection are difficult to distinguish clinically.

The invention discloses a VP (viral protein)0 recombinant protein of DHAV (duck hepatitis A virus)-1 as well as a preparation method and an application of the VP0 recombinant protein and belongs to the technical field of bio-medicine. The preparation method of the VP0 recombinant protein comprises the following steps: a), target fragment cloning of a whole VP0 gene and expression vector construction; b), expression of the VP0 recombinant protein; c), purification of the VP0 recombinant protein. The invention further provides research on the function of the VP0 recombinant protein in detection and immune protection. A novel way is provided for detection, prevention and treatment of duck viral hepatitis, an indirect ELISA (enzyme linked immunosorbent assay) method based on the VP0 recombinant protein is established, and the method has the characteristics of good repeatability and high specificity.

The invention relates to a fluorescence PCR kit for rapidly quantitative detection of gene C-type duck hepatitis A virus. The rapidly qualitative and quantitative detection of the gene C-type duck hepatitis A virus comprises: a) RNA lysate, b) RNA enzyme inhibitor, c) reverse transcriptase, d) reaction solution of the reverse transcription, e) fluorescence quantitative reaction solution, f) SYBRP remix Ex Taq TM II, and g) a standard positive template, wherein the d) the reaction solution of the reverse transcription, e) the fluorescence quantitative reaction solution and f) the SYBRP remix Ex Taq TM II comprises TaKaRa Ex Taq HS, dNTP Mixture, Mg<2+> and SYBRGreen I. The sensitivity of the kit is up to 3.36 x 10<3> copies per reaction and can totally meet requirements of the rapidly quantitative detection of the gene C-type duck hepatitis A virus.

The invention discloses divalent egg yolk antibodies of type I and novel duck hepatitis as well as a preparation method and an application thereof. The preparation method comprises the steps of high-immunity egg collection, high-immunity egg sterilization, fat removal with sodium alginate, precipitation with ammonium sulfate, ultrafiltration membrane concentration and purification of the antibodies, bacteria removal by inactivation and the like. The research results show that the divalent egg yolk antibodies of type I and novel duck hepatitis, which are obtained through grease removal by adopting sodium alginate and precipitation by adopting ammonium sulfate, have high purity, high titers, small side effects and obvious treatment effects and can be used for simultaneously controlling type I and novel duck hepatitis viruses safely and conveniently.

The invention provides preparation methods for duck hepatitis virus immunogen and hyperimmune serum and application of the duck hepatitis virus hyperimmune serum. According to the preparation methods and the application of the duck hepatitis virus hyperimmune serum, the duck hepatitis virus immunogen is obtained through inoculating a serum 1 type duck hepatitis virus CH60 strain DHAV-1 (Duck Hepatitis A Virus type 1) or a serum 3 type duck hepatitis virus CH1 strain DHAV-3 (Duck Hepatitis A Virus type 3) to an allantoic cavity of a chick embryo of 9-10 days old or a duck embryo of 10-12 days old and carrying out proliferation and treatment, and the hyperimmune serum is obtained through mixing the duck hepatitis virus immunogen with a Freund's complete adjuvant or Freund's incomplete adjuvant to prepare solutions of different concentrations, carrying out repeated immunization on immune animals and then sampling and collecting blood and can be applied to the diagnosis and detection on a duck hepatitis virus. The preparation methods provided by the invention have the advantages that the conditional requirements are low, the operation is simple, and the obtained immunogen can meet the requirements on the preparation of specific antiserum.