86 results about "Avian leukemia virus" patented technology

The invention provides a GeXP quick detection kit and detection method for simultaneously identifying 8 chicken infectious immunosuppression disease pathogens. The kit is used on the basis of a GeXP system, and comprises 8 pairs of PCR (polymerase chain reaction) primers. The detection result indicates that the kit has the advantages of high specificity and high sensitivity and can be used for simultaneously identifying and detecting chicken Marek's disease virus, A, B and J subgroup avian leucovirus, avian reticuloendothelium hyperplasia virus, avian reovirus, chicken infectious anemia virus and infectious bursal disease virus.

The invention belongs to the cross technical field of molecular immunology and virology, and particularly relates to a hybridoma cell based on hybridoma type leukosis virus subgroup J isolated strain with preservation number of CGMCC (China General Microbiological Culture Collection) No. 5018, a high-specificity monoclonal antibody of avian leukosis virus subgroup J (ALV-J) surface protein (SU) secreted by the hybridoma cell and a preparation method of the monoclonal antibody. By using the high-specificity monoclonal antibody prepared by the invention, a technical support is provided for diagnosis, prevention and treatment of avian leukosis virus subgroup J disease, and the economic loss caused by avian leukosis virus subgroup J disease is effectively reduced.

The invention discloses an anti-avian leukosis virus p27 protein monoclonal antibody, a gold-colloidal strip containing the same, and application. The anti-avian leukosis virus p27 protein monoclonal antibody is generated by secretion of a hybridoma cell strain with a preservation number of CGMCC NO. 11089 or a hybridoma cell strain with a preservation number of CGMCC NO. 11090. The monoclonal antibody is high in titer to p27 protein and good in specificity, and therefore can be used for preparing a kit and the gold-colloidal strip for detecting an avian leukosis virus. A preparation method of the monoclonal antibody provided by the invention is simple, and an antibody purification process is simple, and the efficiency is high, and the cost is low. As the gold-colloidal strip prepared by the anti-avian leukosis virus p27 protein monoclonal antibody provided by the invention is adopted to detect the anti-avian leukosis virus, the specificity is strong, and the operation is simple, and convenience, speediness and simplicity are realized, besides, a special instrument and equipment are not needed, and professional training is not needed, and a result is clear and easy to recognize; the operation is simple and convenient; the popularization is easy, and the antibody is more suitable for real-time detection.

The invention belongs to the technical field of animal epidemic disease serological diagnosis, and relates to an enzyme-linked immunosorbent assay vector and a kit for detecting avian leukosis P27. The avian leukemia virus (P27) enzyme linked immunosorbent assay kit comprises a 96 hole elisa plate coating a avian leukosis P27 polyclonal antibody, P27 monoclonal antibody marked by alkaline phosphatase, a substrate solution, a stop solution and a cleaning solution. The vian leukosis P27 polyclonal antibody and the P27 monoclonal antibody marked by alkaline phosphatase effectively improve the sensitivity, the specificity and the stability of the detection. The invention provides an efficient and sensitive ELISA (enzyme linked immunosorbent assay) detection kit for sifting out and purifying avian leukosis positive chickens and cultivating new species with heredity resistance, and the kit is low in cost and simple and convenient to operate, and is applicable to promotion in animal husbandry.

The invention discloses multiplex PCR (polymerase chain reaction) detection primers for avian leukosis viruses. The primers comprise detection primers for detecting subgroup A, subgroup B and subgroup J of avian leukosis viruses. The inventional also discloses a reaction system and reaction conditions of multiplex PCR. The detection method provided by the invention has the advantages of being rapid, accurate and cheap.

The invention discloses establishment of a PCR-HRM analysis method for rapid differential diagnosis of different serotypes of avian leukemia viruses. According to the invention, universal primers are good in degeneracy, have very good amplification performance to the A, B, C, D and E subtypes of avian leukemia viruses, is conductive to improving the efficiency of PCR (polymerase chain reaction), and reduces the time of identification and typing of viruses; and specific primers provided by the invention are good in specificity, and the J subtype can be amplified in a specific way. HRM analysis is performed on the amplification product obtained by using the primers, so that the typing of avian leukemia viruses can be performed accurately and rapidly in a high-throughput way by being compared with the standard HRM curve of a known subtype, particularly the endogenous type of avian leukemia viruses and the exogenous type of avian leukemia viruses can be rapidly and accurately distinguished.

The invention discloses an LAMP (Loop-Mediated Isothermal Amplification) kit for detecting a main subtype avian leukemia virus. A loop-mediated isothermal amplification (LAMP) technology is adopted, and two pairs of specific primers (inner primers FIP and BIP, outer primers F3 and B3) are designed according to a POL (Point Of Load) gene sequence of the avian leukemia virus. By applying the LAMP kit and a detecting method established by the LAMP kit, an LAMP real-time turbidity meter is utilized to carry out real-time, quantitative and whole-course sealed monitoring analysis on LAMP reaction primers of the ALV (Avian Leukemia Virus), a reaction system and a reaction process, so that LAMP primers of the ALV are efficiently and specifically amplified. The LAMP kit disclosed by the invention has the advantages that the specificity is strong, the sensitivity is high, a result is quickly obtained, pollution is not caused and a product can be detected in real time; the avian leukemia virus can be detected out in real time by sampling according to the established system, and the detected result can be quickly and accurately obtained, so that convenience is brought for simply and quickly detecting the avian leukemia virus.

The invention belongs to the field of biological technical detection and particularly relates to an immune PCR reagent kit for detecting avian leukemia virus. The reagent kit comprises two antibody oligonucleotide probes, a coupled reaction solution, DNA ligase, protease, a Fast Master mixed solution and a Universal q PRC reaction solution. The immune PCR reagent kit uses a biotin-labeled avian leukemia virus preventing special antibody and a commercialized ortho-position connecting reagent, can detect avian leukemia virus antigen through immune PCR and can quickly detect the avian leukemia virus antigen in a high-throughout mode through the immune PCR without carrying out a complex nucleic acid extracting process on a sample. The immune PCR reagent kit for detecting the avian leukemia virus can be applied to clinical detection and purification of avian leukemia virus and will fill domestic and oversea correlated technique blank.

The invention belongs to the technical field of immunoassay and bio-sensing, and discloses a preparation method and an application of an avian leukemia virus antigen immunosensor which is used for quickly detecting the avian leukemia virus antigen. According to the manufacturing scheme adopted by the invention, the preparation method comprises the following steps: by taking a glassy carbon electrode as a working electrode, modifying beta-CD / MWCNTs; then, adding adamantanecarboxylic acid / capturing antibody Ab1, bovine serum albumin, avian leukemia virus antigen as well as beta-CD@Fe3O4@SiO2-Fc / Ab2 solution in sequence. The beta-CD@Fe3O4@SiO2-Fc is easy to separate due to the superparamagnetism of a ferric oxide nano material; by utilizing the supramolecular recognition characteristic of the cyclodextrin, a great deal of ferrocenecarboxylic acid Fc can be captured through the action of subjective and objective objects, the biocompatibility and the water solubility are good, so that the preparation method is beneficial for increasing the antibody detecting amount, capable of realizing relatively high sensitivity and lowering the detecting limit to 0.33pg / mL.

The invention discloses a typing method of resistance for resisting a subgroup A avian leukosis virus by quality chickens, which adopts an SNP locus at the 619th site of a TVA gene sequence as a research object; PCR-RLFP analysis is performed to detect whether mutation occurs at the SNP locus, and also to detect whether 4 bases are inserted into SNP loci at the 305th-308th sites of the TVA gene sequence; the two detection results are combined together to determine whether the detected chicken is a genetic resistance type or a genetic susceptibility type. The resistance typing method of the invention performs detection from the source, and omits troubles of later-stage detection; the detection method is rapid, accurate, strong in purposiveness, and simple in operation, has practice significance, and can realize rapid selection of chicken genetic characters and improvement of disease resistance of strains.

The invention relates to a J subset avian leukosis virus rapid detection test paper card which comprises a lining board; a sample pad, a golden-standard pad, a cellulose nitrate membrane and a water-absorbing pad are disposed on the lining board; the invention is characterized in that the golden-standard pad is coated with colloidal gold labelled monoclonal antibodies of mouse anti-J subset avian leukosis virus gp85 protein; the cellulose nitrate membrane is coated with a detection line formed by rabbit anti-J subset avian leukosis virus polyclonal antibodies and a quality control line formed by rabbit anti-mouse IgG polyclonal antibodies. The advantages of the invention are that: the detection method of the J subset avian leukosis virus rapid detection test paper card of the invention can rapidly detect whether a chicken body is infected by or carries J subset avian leukosis viruses, and has the advantages of rapid reaction, high sensitivity, strong specificity, simple operations, suitability for near-the-pen detection, economy and practicality.

The invention discloses a K subgroup avian leukosis virus detection kit, which relates to the technical field of virus fluorescent quantitative PCR detection. The kit of the invention comprises one pair of specific primers, and nucleotide sequences of the specific primers are respectively as shown by SEQ ID No. 2 and 3. The kit of the invention is simple in application method, low in cost, easy inobserving a reaction result, high in sensitivity, high in specificity, capable of rapidly detecting K subgroup avian leukosis viruses, very suitable for disease monitoring, on-site emergency and clinical sample detection and suitable for large-scale popularization and application.

The invention relates to a method for diagnosing avian leukosis virus subgroup J of sicken chicken flocks and belongs to the technical field of molecular biology. According to the method, C type retrovirus characteristics of the avian leukosis virus subgroup J are taken as a breakthrough point, on the theoretical basis of identifying a JL-2 strain of the avian leukosis virus subgroup J, specific primers are utilized to clone gp85 fragments of the virus strain, an expression vector PGEX-6P-1 / gp85 category is constructed, an RFLP method is used to detect the genotype of NRAMP1 genes, the enzyme digestion reaction of the fragments where PCR amplification sites are located is observed, according to the research, it not only is indicated that that cocks carrying T allele have higher resistance to avian leukosis virus subgroup J, but also is found that the specificity diagnosis method established by gp85 is a powerful technical means for detecting specificity of ALV-J and timely purifying populations.

The invention discloses a subgroup A or B avian leukosis virus gp85 gene recombinant prokaryotic expression protein and a purification method thereof, and applications. The method includes the following steps: (1) designing and synthetizing specific primers for amplifying gp85 genes; (2) obtaining gp85 gene fragments through PCR amplification, and performing purification; (3) separately cloning the gp85 genes of an ALV-A / B to pET-32a prokaryotic expression vectors, and performing sequencing and identification after screening positive clones; (4) recombining the expression of expression vectorpET-32a-A / B-gp85; (5) purifying expressed gp85 proteins through a urea method; and (6) recombining the renaturation of the gp85 proteins. Thus, soluble gp85 proteins with high purity can be obtained by using the method; and the expressed proteins can be taken as envelope antigens to establish an antibody detection method and used for preparing antiserum for immunizing animals, and also can provideraw materials for immunofluorescence, immunohistochemistry, etc.

The invention provides a reagent kit for detecting avian leukemia virus J sub-groups (ALV-J), and belongs to the technical field of RT-PCR (reverse transcription-polymerase chain reaction) detection. The reagent kit contains a pair of specific primers. Nucleotide sequences of the specific primers are respectively shown as SEQ ID NO.1-2. The invention further provides a method for detecting the avian leukemia virus J sub-groups. The reagent kit and the method have the advantages of high specificity, sensitivity and efficiency, good universality and low cost. Besides, quick differential diagnosis can be carried out on clinical disease samples in 6.5 h, technical means can be provided for early quick diagnosis on the ALV-J and conduction of molecular epidemiological investigation, and accordingly avian leukemia prevention and control can be effectively guided during poultry raising production.

The invention discloses an infectious cDNA (complementary deoxyribonucleic acid) clone, a construction method and application of a recombinant subgroup J avian leucosis virus capable of expressing EGFP (enhanced green fluorescent protein). The infectious cDNA clone has a nucleotide sequence shown by SED ID NO.1. The infectious cDNA clone of the recombinant subgroup J avian leucosis virus capable of expressing the EGFP can be used for successfully rescuing viruses. Moreover, the rescued recombinant subgroup J avian leucosis virus capable of expressing the EGFP can not influence the replication of the virus after the insertion of extraneous source EGFP, overcomes the complexity in the conventional process of detecting the an ALV-J neutralizing antibody at the same time, and can be used for large-scale detection of the ALV-J neutralizing antibody more simply and effectively.

The invention discloses a recombined chicken Marek's disease virus vaccine strain for expressing Gag and Env genes of an avian leukosis virus subgroup J (ALV-J) and a construction method and application of the recombined chicken Marek's disease virus vaccine strain, and belongs to the technical field of medicine or veterinary medicine. By means of the recombination and clone technology, a gene segment CAG-ALVGE containing the ALV-J Gag and Env genes and a CAG promoter sequence is inserted in a US2 gene of the strain 814 of the chicken Marek's disease virus, a recombined cosmid with a CAG-ALVGE expression frame inserted in a US2 gene is constructed, and the recombined chicken Marek's disease virus vaccine strain for expressing the Gag and Env genes is obtained through salvation of the recombined cosmid. Research shows that the obtained vaccine strain has the same in-vitro replication ability as a parent virulent vaccine strain 814 and good hereditary stability, and can resist attacks of a very virulent MDV strain and a very virulent ALV-J strain at the same time. It can be seen that the obtained recombined MDV vaccine strain can be used for preparing medicine for preventing or treating avian leukosis and the chicken Marek's disease.

The present invention provides primers and a detection kit for avian leukosis J subgroup virus PCR detection. According to the present invention, PCR detection primers are designed by analyzing avian leukosis J subgroup virus env gene, wherein nucleotide sequences of the primers are represented by sequence lists SEQ ID No.1 and SEQ ID No.2; and the primers have good specificity, and the detection method has characteristics of rapidness, simpleness, high accuracy and good sensitivity so as to provide an excellent detection method for avian leukosis J subgroup virus identification. The present invention further provides a detection kit for the avian leukosis virus, wherein the detection kit comprises a primer pair represented by the sequence lists SEQ ID No.1-2.

The invention discloses a test strip used in one-step rapid detection of Marek's disease virus (MDV) and subgroup-J avian leukosis virus (ALV-J). The test strip comprises a non-absorbing supporting layer, and an absorption layer adhered to the supporting layer. The absorption layer is formed by sequentially spliced components of an absorption fiber layer, a gold-labeled antibody fiber layer, a cellulose film layer, and a water absorption layer. The cellulose film layer is marked with anti-goat IgG or anti-mouse IgG control blot, and a detection blot comprising anti-MDV and anti-ALV-J antibody. The anti-MDV and anti-ALV-J antibody is an anti-MDV and anti-ALV-J antibody monoclonal or polyclonal antibody. An anti-MDV and anti-ALV-J mixed polyclonal antibody or monoclonal antibody marked by colloidal gold and corresponding to the detection blot is adhered to the gold-labeled antibody fiber layer. The test strip provided by the invention has the advantages of high detection specificity, high sensitivity, simple operation, fast detection, and intuitive result. The test strip is suitable for MDV and ALV-J virus on-site rapid combined detection, and can be used in identification and diagnosis of single infection or mixed infection of the two viruses. The test strip can be widely applied, and is suitable for popularization.

The invention relates to a J substock lymphoid leuoosis-resistant polyclonal antibody and a preparation method thereof. The J substock lymphoid leuoosis-resistant polyclonal antibody can effectively inhibit the infection of J substock avian leukosis virus, and is obtained on the basis of highly conserved sequence synthesized small peptides formed by 27 amino acid in transmembrane protein coded with J substock avian leukosis virus gp 37 gene through a coupled keyhole hemocyanin immune rabbit; and the polyclonal antibody blocks the invasive J substock avian leukosis virus reproduction by being combined with the nucleus non-specifity, and is combined with and eliminates the specificity of the J substock avian leukosis virus, thereby effectively preventing the generation and spreading of the J substock lymphoid leuoosis, greatly reducing the economic loss of aviculture caused by infection of J substock avian leukosis virus, and providing a novel method and thinking for the lymphoid leuoosis preventing and controlling.

The invention discloses a test strip used in one-step rapid detection of reticuendotheliosis virus (REV) and subgroup-J avian leukosis virus (ALV-J). The test strip comprises a non-absorbing supporting layer, and an absorption layer adhered to the supporting layer. The absorption layer is formed by sequentially spliced components of an absorption fiber layer, a gold-labeled antibody fiber layer, a cellulose film layer, and a water absorption layer. The cellulose film layer is marked with anti-goat IgG or anti-mouse IgG control blot, and a detection blot comprising anti-REV and anti-ALV-J antibody. The anti-REV and anti-ALV-J antibody is an anti-REV and anti-ALV-J antibody monoclonal or polyclonal antibody. An anti-REV and anti-ALV-J mixed polyclonal antibody or monoclonal antibody marked by colloidal gold and corresponding to the detection blot is adhered to the gold-labeled antibody fiber layer. The test strip provided by the invention has the advantages of high detection specificity, high sensitivity, simple operation, fast detection, and intuitive result. The test strip is suitable for REV and ALV-J virus on-site rapid combined detection, and can be used in identification and diagnosis of single infection or mixed infection of the two viruses. The test strip can be widely applied, and is suitable for popularization.

The invention discloses a siRNA recombinant interference carrier based on an ALV-J gp85 gene conserved region as a target sequence, and a corresponding preparation method of the siRNA recombinant interference carrier. In the invention, a gp85 gene is used for designing and synthesizing siRNA to build a hairpin-shaped structure forming the siRNA; further an annealed double-chain DNA is obtained, and then is cloned into an adenovirus carrier pHBAd-U6-RFP to build a recombinant adenovirus carrier pAd-gp85-shRNA2; and after sequencing identification is built successfully, recombinant adenovirus carrier is subjected to homologous recombination with framework plasmids in HEK 293 cells. Experiments show that the carrier can effectively inhibit the replication of GX13HG03 virus in DF-1 cells, and can play a certain preventive and therapeutic effect on ALV-J in chickens. Therefore, the invention provides an effective adjunct means for the purification of J subgroup avian leukemia virus, thus providing a new way for AL prevention and control and antiviral research.

The invention discloses a ubiquitin-mediated recombinant plasmid expressing avian leukosis virus subgroup J gp85, p27 and p10 genes, and the recombinant plasmid consists of avian leukosis virus subgroup J gp85, p27 and p10 genes, chicken ubiquitin Ub gene and eukaryotic expression vector pVAX1. The corresponding construction method is also established and is capable of realizing serial connection of avian leukosis virus subgroup J gp85, p27 and p10 genes and chicken ubiquitin gene and insertion into the vector pVAX1. An in-vitro experiment proves that the recombinant plasmid is successfully expressed after being used to transfect a DF-1 cell. During a chicken immunity experiment, the antibody positive conversion rate of immunized chicken and antibody positive rate of offsprings are both raised. By constructing the ubiquitin-mediated avian leukosis virus subgroup J multigenic co-expression DNA vaccine, the method helps to establish a basis for researching a novel vaccine for avian leukosis virus subgroup J, and provide an effective auxiliary means for purifying avian leukosis virus subgroup J virus.

The embodiment of the invention discloses a colloidal gold detection kit for detecting avian leukosis virus and application thereof. The colloidal gold detection kit comprises a detection test strip and a micropore reagent, wherein the detection test strip comprises a nitrocellulose membrane; the nitrocellulose membrane is provided with a detection line and a quality control line; the detection line is coated by a monoclonal antibody 1A1 resisting p27 protein of avian leukosis virus; the micropore reagent is a micropore containing the colloidal gold-marked monoclonal antibody 5F1 resisting thep27 protein of the avian leukosis virus. The colloidal gold detection kit has the advantages that the sensitivity in detection is high, the consumption time is short, and the detection efficiency ishigh; the operation is simple, the difficulty in operation is low, the cost is low, and the colloidal gold detection kit is suitable for being popularized and applied to the detection laboratory at base level.

The invention relates to the technical field of biology and in particular relates to a kit for simultaneously detecting an avian leukosis virus antibody and a salmonella pullorum antibody. An antigencombination of the kit comprises p27 protein, gp85 protein and GroEL-delta8-1 protein. Avian leukosis virus capsid protein p27, prokaryotic expression protein of envelope protein gp85, and prokaryoticexpression protein of truncated GroEL-delta8-1 of a salmonella pullorum dominant antigen are used as a coating antigen to develop an ELISA (Enzyme-linked Immunosorbent Assay) kit capable of simultaneously detecting the avian leukosis virus antibody and the salmonella pullorum antibody. Compared with a current common kit for independently detecting avian leukosis or pullorum disease, the kit provided by the invention has the effect of simultaneously detecting the avian leukosis or the pullorum disease, and the detection and purification work of the avian leukosis and the pullorum disease can be extremely alleviated.

The invention discloses a hemangioma pathotype subgroup J avian leukosis virus gene and the construction of infectious clone thereof. A full-length genome sequence of a subgroup J avian leukosis virus of the invention is shown as SEQ ID NO:1. Then, the hemangioma pathotype subgroup J avian leukosis virus gene shown as SEQ ID NO:1 is subjected to amplification by adopting a specific primer; and pBlueskript SK (I) is used as a vector and chick embryo fibroblast is used as a host cell to construct the infectious clone of the hemangioma pathotype subgroup J avian leukosis virus gene so as to lay the foundation for subsequent research on relation among pathogenic mechanism, a gene structure and functions, virus reproduction, genetic variation rules and the like of the hemangioma pathotype subgroup J avian leukosis virus ALV-J.

The invention relates to the technical field of molecular biology, in particular to an RT-RAA fluorescence method detection primer pair, a kit and a detection method for a J subtype avian leukosis virus gp85 gene. The primer pair comprises a forward primer and a reverse primer; and the nucleotide sequence of the forward primer is as shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.2. The primer pair can specifically amplify the J subtype avian leukosis virus gp85 gene, the J subtype avian leukosis virus can be specifically detected by utilizing the primer pair, the detection period is shorter, and the specificity is higher.

The invention discloses a purification method for a single virus in avian leukemia virus mixed infection. End point dilution is carried out by measuring TCID50 of mixed inflection virus, DF-1 cells are inoculated by adopting a diluted virus solution, supernatant and a cell culture are collected, and a parting authentication primer and an IFA are used for carrying out authentication, so that a single subtype avian leukemia virus is obtained. The method is simple for experiment condition requirement, and has extremely high practical application values. Experimental results prove that different subtype mixed infection ALVs can be purified successfully, and technical support can be provided for research of avian leukemia virus molecular biology characteristics and pathogenicity thereof.

The invention discloses a method for identifying avian leukosis virus and chicken infectious anemia virus through visual double LAMP. The invention provides a single-stranded DNA group for identifying avian leukosis virus and chicken infectious anemia virus through visual double LAMP. The single-stranded DNA group consists of single-stranded DNA molecules as shown in SEQ ID No.1-10. According to the invention, the visual double LAMP method for identifying and detecting the ALV and the CIAV is successfully established. The double LAMP can identify and diagnose the ALV and the CIAV in the same reaction tube, has the advantages of good specificity, high sensitivity, small pollution, convenience, rapidness and the like, a detection result can be directly observed by naked eyes, the result is judged according to the color of a reaction product, and the double LAMP is suitable for clinical rapid screening of the ALV and the CIAV.

The invention discloses a DF-1 cell culture method used for avian leukemia virus separation. The method comprises the following steps that 1, centrifugation is conducted on collected anticoagulant blood at 1500 rpm for 10 min, and white villiform lymphocytes at the joint between supernatant and sediments are aspirated into a 1.5 mL centrifuge tube to form a blood plasma sample; 2, a DF-1 cell suspension is added into 96-hole cell culture plate, wherein cell culture liquid exists in each hole; 3, the separated blood plasma sample and positive and negative controls are added into each hole respectively; 4, the cell culture plate is placed into a cell culture box for culture for 24 hours, then the cell culture liquid is replaced by cell maintenance liquid, and culture continues; 5, after 9 days, repeated freezing and thawing is conducted on cells at a temperature of 80 DEG C and room temperature for 2-3 times; 6, after the last freezing and thawing, cultures are collected, and an antigenp27 is detected by means of an ELISA. By means of the method, culture of the DF-1 cells is fast, and the culture quality of the cells is good.