145 results about "Tembusu virus" patented technology

The invention discloses a primer pair and a kit for identifying duck adenovirus II type, wherein the nucleotide sequence of the primer pair is represented as the SEQ ID No.1-2. The primer pair is strong in specificity, can accurately and high-effectively identify whether a to-be-test sample contains the duck adenovirus II type or not, while other poultry viruses, comprising poultry adenovirus, parvovirus, Tembusu virus and Newcastle disease virus, cannot be amplified to form DNA fragments. The primer pair is high in sensitivity, can at least detect 0.2258 ng of sample DNA. The primer and the PCR detection kit are short in detection time, wherein the whole PCR process only lasts for about 2 h. The primer pair and the kit are simple and economical, are free of expensive experimental instrument and reagents, can be used for detecting whether ill ducks are infected with the virus or not timely, so that greater economic loss can be avoided by means of corresponding measures.

The invention provides monoclonal antibodies against the duck Tembusu virus, an antigen detection kit and application. According to the invention, the DTMUV-JXSP cell strain of the duck Tembusu virus is used as immunogen for preparation of the monoclonal antibodies which include monoclonal antibodies respectively secreted by hybridoma cells with respective accession numbers of CGMCC No. 8104, CGMCC No. 8107 and CGMCC No. 8106; the monoclonal antibody 3B4 secreted by the hybridoma cell with the accession number of CGMCC No. 8106 is used as a primary antibody coated ELISA plate, the monoclonal antibody 3F2 secreted by the hybridoma cell with the accession number of CGMCC No. 8107 is subjected to enzyme labeling and used as an ELISA secondary antibody, so the double-antibody sandwiched detection kit is established and the kit has good sensitivity and specificity. According to the invention, effective means are provided for large-scale clinical detection of the duck Tembusu virus.

Provided herein is a GeXP detection kit for identification of 11 kinds of duck virus diseases. The detection kit includes a primer set for identifying or auxiliarily identifying pathogens of duck communicable diseases, including one or more of primer pair A, primer pair B primer pair C, primer pair D, primer pair E, primer pair F, primer pair G, primer pair H, primer pair I, primer pair J, primer pair K and primer pair L. The can kit detect, simultaneously avian influenza virus, subtype H5, H7 and H9 of avian influenza virus, duck hepatitis virus, duck enteritis virus, duck Tembusu virus, Newcastle disease virus, egg drop syndrome virus, Muscovy duck reovirus, Muscovy duck parvovirus and duck circovirus with the primer set, PCR reagent or primer pairs provided in the present invention.

The invention relates to a duck tembusu virus (DTMUV) E protein gene, and the amino acid sequence of the DTMUV E protein gene is SEQ ID NO:1. The DTMUV genetic engineering subunit vaccine obtained by the invention is inoculated to DTMUV negative Sheldrake in the age of 20 days by virtue of muscles, blood is sampled and a DTMUV antibody is determined 14 days after inoculation, DTMUV antibodies in an immunization group are all positive, the effective rate reaches up to 100%, and no antibody is detected in a control group. Results show that the prepared recombinant protein has good immunogenicity, and further the prepared DTMUV genetic engineering subunit vaccine can arouse immune response of the Sheldrake.

The invention discloses a one-step SYBR Green fluorescence quantitative PCR detection kit used for detecting duck Tembusu virus. The duck Tembusu virus detection kit comprises two specific primers SEQ1 and SEQ2 designed according to conservative regions of duck Tembusu virus E gene. With the detection kit provided by the invention, operation steps are simplified, reaction time can be shortened, pollution possibility is reduced, and detection accuracy and speed are improved. A detection reaction can be completed within 2 hours. With fluorescence quantitative PCR, after amplification is finished, initial virus copy number can be directly quantified through a standard curve, and reliable information can be provided for epidemiological investigations. Also, the fluorescence quantitative PCR kit provided by the invention has the advantages of simple and fast operation, and relatively low cost. Therefore, the kit can satisfy requirements of field sample testing.

The invention discloses a duck tembusu virus low virulent strain. The low virulent strain is obtained through mutation after continuous subculture of duck tembusu virus virulent strain FX2010 on chicken embryo fibroblast (CEF). The invention further discloses a vaccine for preventing or treating duck tembusu virus disease. The vaccine is duck tembusu virus disease live attenuated vaccine or inactivated vaccine using the duck tembusu virus low virulent strain for vaccination. The invention further discloses a detection antigen for diagnosing the duck tembusu virus disease. The detection antigen is virus particle of the duck tembusu virus low virulent strain. The protection rate of the duck tembusu virus low virulent strain to virulent attack is 100% after the duckling and egg-laying duck are vaccinated whether the duck tembusu virus low virulent strain is used as live attenuated vaccine or the inactivated vaccine; the duck tembusu virus low virulent strain has good specificity as the detection antigen; therefore, the duck tembusu virus low virulent strain has excellent application prospect in the aspect of preventing and treating and diagnosing the duck tembusu virus disease.

The invention belongs to the technical field of detection of animal virology and animal infectious diseases, and particularly discloses a duck tembusu virus E truncated protein and application. Protein of an amino acid sequence as shown in SEQ ID NO. 2 is used as a coating antigen, a duck tembusu virus serum detection kit which is prepared from a hybridoma cell strain under the accession number CCTCC NO: C2017169 is used for detecting duck serum and an egg yolk antibody which are infected by wild type virus, and has good specificity; and the sensitivity is high, and after being diluted to be 1: 12800, duck tembusu virus positive serum can still be positive when detected. Compared with the traditional agar diffusion test, the duck tembusu virus E truncated protein is high in coincidence rate, simple and convenient to operate, short in detection time and suitable for simultaneously detecting a large number of samples.

A monoclonal antibody against the Duck Tembusu virus and a hybridoma cell line secreting the monoclonal antibody, a reagent kit and method for detecting a Duck Tembusu virus antibody, and application of the monoclonal antibody in preparing products for diagnosing the Duck Tembusu virus disease. The monoclonal antibody may bind specifically to E protein of Duck Tembusu virus and has an activity of neutralizing Duck Tembusu virus.

The invention relates to the field of animal prevention medicine, particularly to a preparation method for an injection for preventing animal epidemic diseases from occurring, wherein the propolis injection for preventing duck hemorrhagic oophoritis is composed of a propolis aqueous solution, a duck embryo homogenated tissue inoculated with duck flavivirus-Tembusu virus and dead by infection, andan inactivation solution; the preparation method comprises the step of adding the inactivation solution in the mixed solution of the duck embryo homogenated tissue and propolis to obtain the propolisinjection for preventing duck hemorrhagic oophoritis; the propolis injection for preventing duck hemorrhagic oophoritis prepared by the preparation method of the invention has a remarkable effect to prevent duck hemorrhagic oophoritis (duck Tembusu virus disease); the protection ratio is increased to 90-100%, the protection period is prolonged to 6-12 months, the action time is shortened to 5 days, and only once injection can achieve an expected effect.

Belonging to the field of traditional Chinese medicines, the invention particularly relates to a Chinese medicinal composition treating a duck Tembusu virus. The Chinese medicinal composition is prepared from the following raw materials by weight: 45g of Schizonepeta, 30g of radix sileris, 25g of notopterygium roots, 25g of radix angelicae biseratae, 30g of radix bupleuri, 25g of Radix Peucedani, 30g of fructus aurantii, 45g of Poria cocos, 30g of Platycodon grandiflorum, 25g of Ligusticum wallichii, 15g of liquorice, 15g of mint, 30-50g of Venenum Bufonis, 40-80g of radix isatidis, 50-100g of folium artemisiae argyi, 30-50g of Polygonum multiflorum, 40-80g of hawthorn, and 40-80g of dried tangerine peel. The Chinese medicinal composition provided in the invention has very good efficacy on poultry egg laying decrease, inappetence, depression and diarrhea caused by the duck Tembusu virus, an influenza virus and an egg drop syndrome virus, and especially has significant efficacy on sudden egg drop syndrome caused by duck Tembusu virus infection.

The invention discloses a double RT-PCR detection kit for H9 subtype avian influenza virus and duck tembusu virus. The kit comprises two pairs of specific primers. The detection kit disclosed by the invention has the advantages of simplicity in operation, high sensitivity, high specificity, good repeatability and the like; by adopting the detection kit disclosed by the invention, a double PCR detection method for H9 subtype avian influenza virus and duck tembusu virus can be established, which detects and identifies the two pathogens of H9 subtype avian influenza virus and duck tembusu virus at the same time and is applied to the mixed infection of duck tembusu virus resulting from the hypoimmunity caused by the H9 subtype avian influenza virus infection; therefore, the time cost can be saved, and the pollution can be reduced.

The invention discloses a multiplex fluorescence quantitative PCR detection primer and probe combination and detection method for simultaneously detecting four duck-susceptibility viruses. Sequences of primers include SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:7, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:11 respectively; sequences of probes include SEQ IDNO:3, SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO:12 respectively. The four duck-susceptibility viruses include the duck-derived avian influenza virus, the duck-derived newcastle disease virus, the duck hepatitis a virus and the duck tembusu virus. By means of the multiplex fluorescence quantitative PCR detection primer and probe combination and detection method, the four viruses can be detected simultaneously, pathogens can bequantitatively detected, the detection is quick and convenient, and the time and labor are saved.

The invention discloses a triple fluorogenic quantitative PCR detection primer, kit and method of the duck tembusu virus disease, the egg drop syndrome virus and the H9 subtype avian influenza virus. The detection primer for the duck tembusu virus disease, the egg drop syndrome virus and the H9 subtype avian influenza virus is shown in SEQ ID NO.1-6. The detection primer and kit can detect the EDSV, the H9 AIV and the DTMUV at the same time. No cross reaction with duck plague virus, goose parovovirus, muscovy duck parvovirus and escherichia coli genomic DNA and duck hepatitis A virus 1, duck hepatitis A virus 3, muscovy duck reovirus and newcastle disease virus RNA exists. The lower limits of detection for EDSV nucleic acid, H9 AIV nucleic acid and DTMUV nucleic acid are 8.0 copies, 4.8 copies and 1.3 copies respectively. The detection primer, kit and method can be used for qualitative and quantitative detection for the EDSV, the H9 AIV and the DTMUV.

The invention provides a multiplex PCR detection primer and method for important viruses causing egg-laying abnormality. The method comprises the steps of preparing specificity capture probes aiming at viral nucleic acids of three important viruses such as an egg-laying descending syndrome virus, an avian Tembusu virus and an H9 subtype avian influenza virus which cause the egg-laying abnormality, carrying out hybridization on the specificity capture probes and the corresponding viral nucleic acids, then carrying out cyclization in the presence of ligase, carrying out PCR amplification by virtue of a pair of universal detection primers specific to the probes so as to detect, and determining infection pathogeny according to the size of an obtained amplified product. The method can be used for detecting a single virus and simultaneously differentially diagnosing three different viruses, has the characteristics of high efficiency, specificity and sensitivity and low cost, is applicable to the detection analysis of a large number of clinical samples when laying fowl cannot normally lay eggs, and is an important technological measure for the early rapid differential diagnosis and the molecular epidemiological analysis of pathogeny which definitely causes the egg-laying abnormality.

The invention discloses a method and corresponding primer and probe for distinguishing duck tembusu virus wild virulent strain and vaccine strains and corresponding primer and probe. According to themethod, a fluorescent PCR (Polymerase Chain Reaction) detection method capable of quickly distinguishing the duck tembusu virus wild virulent strain and vaccine strains (a WF100 strain and an FX2010-180P strain) is established for the first time; the detection method is simple to operate; all the whole operation processes does not exceed 3 hours; the time needed by the identification and detectionof the virus wild virulent strain and vaccine strains is obviously shortened, and moreover, the method is high in accuracy, good in specificity and good in repeatability, can be used for carrying outanalysis in an accurate, quick and high-throughput manner, and is beneficial to the popularization and application in clinical practice.

The invention discloses a method for preparing a duck tembusu virus inactivated vaccine and the duck tembusu virus inactivated vaccine. A cell line used for virus inoculation is EB66 cell line (a duckembryonic stem cell-derived cell strain), and a bioreactor is adopted for virus culture in a serum-free full suspension manner; and the method comprises the following steps: 1) breeding of virus species; 2) establishment of virus seed batches; 3) preparation of cell venom; 4) inactivation of viruses; and 5) emulsification and other steps to complete the preparation of the vaccine. The method provided by the invention has the characteristics that the prepared cell venom is high in virus content, the production process is stable, intelligent control is achieved, large-scale serum-free suspension culture is realized, the operation is easy, and the cost is low; and the prepared duck tembusu virus inactivated vaccine has the advantages of safety, low side reactions, high immune efficacy, smallbatch-to-batch difference, small number of tests, low cost and the like, and is an ideal vaccine for preventing the occurrence and the prevalence of a duck tembusu virus disease in the waterfowl industry.

The invention provides a recombinant duck viral enteritis virus vaccine for expressing secretory duck Tembusu virus M / E protein, and a construction method and application thereof. The collection number is CCTCC NO:V201215, and the name is rDEV-TME-tPAS. By using a recombinant clone technique, a gene segment rDEV-TME-tPAS comprising an SV40 promoter, duck Tembusu virus M and E proteins and a tPA (Tissue plasminogen activator) signal peptide sequence is inserted into a spacer between the US7 and US8 genes of the duck viral enteritis virus to construct a cosmid in which the rDEV-TME-tPAS expression frame is inserted between the US7 and US8 genes, thereby obtaining the recombinant duck viral enteritis virus vaccine CCTCC V201215 for expressing secretory duck Tembusu virus M / E protein. The invention also provides a method for constructing a recombinant duck viral enteritis virus vaccine strain and application of the recombinant duck viral enteritis virus vaccine strain in preparing a vaccine for preventing infectious diseases caused by duck viral enteritis virus and duck Tembusu virus.

The invention discloses a preparation method for expressing the duck Tembusu virus (DTMUV) prm and the E protein recombinant Newcastle disease virus (NDV). The preparation method comprises the following steps: 1) constructing full-length plasmids of an attenuated ND GM strain; 2) constructing plasmids for expressing the DTMUV prm and the E protein recombinant NDV; 3) rescuing and identifying the recombinant virus aGM-prm / E. The invention also discloses the virus aGM-prm / E prepared by the method. The virus aGM-prm / E is collected at the China Center for Type Culture Collection (CCTCC), with collection number of CCTCC V201644. A vaccine prepared by utilizing the virus can prevent the ND and the DTMUV disease and conduce to reducing virus removal after virulent NDV infection.

The invention discloses a duck tembusu virus (DTMUV) infectious clone attenuated vaccine strain. The attenuated vaccine strain is a clone strain of a parent virus DTMUV attenuated vaccine strain FX2010-180P and has a whole-genome sequence shown as SEQ ID NO.1. The infectious clone attenuated vaccine strain is obtained through a reverse genetic operation method. The invention further discloses a preparation method and application of the DTMUV infectious clone attenuated vaccine strain. The DTMUV infectious clone attenuated vaccine strain not only can be used for developing a novel DTMUV disease vaccine but also can be used as a virus vector for expression of exogenous genes, and meanwhile the strain is an important tool for DTMUV molecular biology study.

The invention discloses a duck tembusu virus envelop E protein inhibitory peptide and application thereof, belonging to the technical field of animal virus molecular biologics. DTMUV envelop E protein is obtained by using a genetic engineering method, DTMUV HN1 strain E protein is taken as a target, the duck tembusu virus envelop E protein inhibitory peptide is obtained through screening by using a bacteriophage random 12 peptide library, and the amino acid sequence of the duck tembusu virus envelop E protein inhibitory peptide is as shown in HWSTRQGSTRWN. By adopting the inhibitory peptide, proliferation of the duck tembusu virus in duck embryo fibroblast can be inhibited, the virus copy number of the DTMUV in the duck embryo fibroblast can be remarkably reduced in different concentrations, meanwhile the virus titer of the DTMUV in the duck embryo fibroblast can be remarkably reduced, and the DTMUV has no remarkable influence on proliferation of the duck embryo fibroblast. The duck tembusu virus envelop E protein inhibitory peptide has good application prospect in preparing medicines or feed additives for preventing the duck tembusu virus, and can be used for preventing and controlling the duck tembusu virus.

The invention discloses a preparation method of a reporter duck tembusu virus and a product and application thereof. The method includes: modifying 1st -2646th nucleic acid segments of a duck tembusuvirus genome through a reverse genetics operation system of a CQW1 strain (GenBank: KM233707.1), and inserting NanoLuc reporter gene to space between 5'UTR of TMUV virus and structural gene to build full-length cDNA infectious clone of reporter virus. By optimizing building of the reporter virus and replacing the reporter gene, passage stability of the reporter virus is improved effectively, and the reporter virus after being saved does not lose the reporter gene within 5 generations; the number of primers and amplified gene segments used for building recombinant plasmid is reduced, reaction steps are reduced, process is simplified, convenience is brought to operation, period for obtaining the reporter virus is shortened effectively, and production cost is lowered.

The invention discloses a tembusu virus nano PCR (Polymerase Chain Reaction) detection kit and a detection method thereof; the kit comprises a 5* reverse transcription buffer solution, a dNTP Mixture, reverse transcriptase, a RNA enzyme inhibitor and a reverse transcription primer and is characterized by further comprising 2*NanoPCR Mix, an upstream primer and a downstream primer, wherein the sequence of the upstream primer is represented by SEQ ID No.1; and the sequence of the downstream primer is represented by SEQ ID No.2. The kit can be used for detecting tembusu virus. The invention further provides the detection method of tembusu virus by adopting the kit. Tembusu virus can be detected rapidly and specifically; and therefore, the detection efficiency and the specific amplification yield of tembusu virus can be greatly increased.

The invention discloses a kit for identifying virulent strain and attenuated vaccine strain of duck tembusu virus and a use thereof. The kit comprises an upstream primer P1 aiming at a duck tembusu virus virulent strain sequence shown in the formula of SEQ ID NO: 1, wherein the last basic group at the tail end 3' of the P1 is the same as a 1454th basic group of the sequence shown in the formula of SEQ ID NO: 1 and the penultimate basic group at the tail end 3' of the P1 is the same as a 1453th basic group of the sequence shown in the formula of SEQ ID NO: 1, also comprises an upstream primer P2 aiming at a duck tembusu virus attenuated vaccine strain sequence shown in the formula of SEQ ID NO: 2, wherein the penultimate basic group at the tail end 3' of the P2 is the same as a 1891th basic group of the sequence shown in the formula of SEQ ID NO: 2, and also comprises an universal downstream primer P3 aiming at a 1971th-10991th nucleotide sequence shown in the formula of SEQ ID NO: 1 or SEQ ID NO: 2. The kit can fast, simply and accurately distinguish natural-infection virulent strain and attenuated vaccine strain of duck tembusu virus, has high specificity, high sensitivity and good repeatability and has a very important meaning for clinical wide application of the attenuated vaccine strain of duck tembusu virus.

The invention aims to provide a duck Tembusu virus gene engineering subunit vaccine which is prepared by the following steps: screening to obtain duck Tembusu virus E protein with Domain III structure field of dominant antigen epitope, constituting a novel fusion protein LTB-Es from Lingker and enterotoxin LTB, and preparing the duck Tembusu virus gene engineering subunit vaccine by using the fusion protein as the antigen. The amino acid sequence of the coding protein of the duck Tembusu virus novel fusion protein LTB-Es is SEQ ID NO:1, and one of the nucleotide sequences is SEQ ID NO:2. The prokaryotic expression vector is utilized to construct the Escherichia coli BL21(DE3) host bacterium capable of expressing the duck Tembusu virus novel fusion protein LTB-Es. The gene engineering subunit vaccine prepared from the purified recombinant expressed protein can enable the immunized duck group to obtain immunoprotection.

The invention discloses a process method of extracting an egg yolk antibody of a duck tembusu virus. The process method comprises the following steps of: S1, directly mixing acidified water with a yolk original liquid; breaking an emulsifying state under the action of mechanical stirring and water, wherein in the pH environment, the solubleness of the egg yolk antibody is the minimum and non water soluble lipoproteins are precipitated, and oil and an aqueous solution are separated to form a precipitated; S2, adding a proper amount of diatomite into the aqueous solution extracted in the first step for micro-filtration, wherein in acidic condition, the content of proteins is almost while the settling amount of lipoproteins is relatively great; and S3, mixing the aqueous solution extracted in the step 2 with caprylic acid, wherein caprylic acid is reacted with impure proteins in the aqueous solution to form large-particle substances which are separated from an immune globulin aqueous solution to achieve a purpose of extracting the immune globulins. The method disclosed by the invention has the advantages of being high in antibody recovery rate, simple to operate and the like.

The invention discloses a preparation method of a refined yolk antibody against duck tembusu virus. The preparation method comprises the following steps: antigen preparation; preparation of a duck tembusu virus transfer factor-interleukin 2 adjuvant inactivated vaccine; production of a high-immunity egg; disinfection; yolk separation; antibody extraction; and filtering to obtain a finished product. In the yolk antibody prepared by the preparation method disclosed by the invention, when the neutralizing antibody titer in the duck tembusu virus yolk is greater than or equal to 1:512, the ducks are medicated once; 2 days later, the death can be controlled, and 3 days later, normal feed intake can be recovered, and clinical disease symptoms disappear; in combination of antibiotics, the effect of controlling secondary infection of bacteria is better; the prognosis development of commercial ducklings is sound, and the economic value is not influenced; with timely treatment of commercial laying ducks and breeding ducks, the laying rate is improved by 5-13% on average after the feed intake recovers to normal for 7 days; and the refined yolk antibody against duck tembusu virus provides an effective means for clinical treatment of duck tembusu virus.

The invention relates to a duck tembusu virus (DTMUV) disease immunotherapy preparation and preparation method thereof, which belong to the field of bio-pharmaceuticals. The preparation method of the DTMUV disease immunotherapy preparation comprises the following steps of: culturing seed viruses for producing a tembusu virus strain on a large scale, and harvesting a virus liquid; deactivating the collected virus liquid, concentrating, adding the deactivated and concentrated virus liquid into an adjuvant, and emulsifying to obtain an immune antigen; immunizing laying ducks by using the immune antigen, randomly collecting blood and separating serum after last immunization, and measuring the valence of an anti-DTMUV antigen through an Agar Gel Precipiti (AGP) test till the valance of the antigen is qualified; and collecting eggs laid by an immunized qualified duck group, and preparing into a high-immunity egg yolk antibody with the conventional method. The DTMUV disease immunotherapy preparation contains a DTMUV high-immunity egg yolk antibody. The DTMUV disease immunotherapy preparation prepared with the method has the advantages of good treatment effect, safety, reliability and low cost, and is suitable for large-scale industrial production.

The invention discloses a recombinant duck plague virus of expressing duck tembusu virus E protein as well as a construction method and application of the recombinant duck plague virus, wherein the gene of the duck tembusu virus E protein is interpolated inside the genome of the recombinant duck plague virus; and the nucleotide sequence of the duck tembusu virus E protein gene is as shown in SEQ ID NO. 9. The construction method comprises the following steps: (1) substituting the CMV promoter of gfp gene in pDEV-vac with an EF1 promoter so as to obtain pDEV-EF1; (2) interpolating a Pcmv-E-BGH-pA expression cassette into the pDEV-EF1 so as to obtain pDEV-E; and (3) transfecting the pDEV-E with chicken embryo fibroblasts and rescuing so as to obtain the recombinant duck plague virus. The recombinant duck plague virus, compared with a parent strain, has no significant difference in the size of virus plaque, showing that the diffusion of the duck plague virus on the chicken embryo fibroblasts is not affected by the interpolation of the duck tembusu virus E protein gene. After transfecting with the chicken embryo fibroblasts, the recombinant duck plague virus can successfully express E protein, so as to lay a foundation for developing duck plague virus-duck tembusu virus bivalent vaccine.