174 results about "Avian infectious bronchitis virus" patented technology

The invention discloses a infectivity bronchitis attenuated vaccine strain LDT3-A strain, and discloses application and application effect thereof in preventing and curing chicken infectivity bronchitis. The microorganism accession number of the attenuated vaccine strain is CGMCC-2902. The attenuated vaccine strain of the present invention has good safety and good immunization protection effect to the chicken infectivity bronchitis. The attenuated strain can be prepared into single vaccine or combined vaccine for preventing or curing infectivity bronchitis virus.

The method relates to an attenuated vaccine strain for avian infectious bronchitis virus and application thereof. According to the invention, avian infectious bronchitis virus (IBV) is attenuated, so as to successfully obtain an IBV attenuated strain CCTCC.V201232 and a derivative virus strain thereof. The attenuated strain and derivative virus strain provided by the invention can be used in preparation of a vaccine composition for prevention of infectious bronchitis. Experiments show that the attenuated strain and vaccine composition provided by the invention can be inoculated to immature birds, so as to effectively activate immune system in the birds and well prevent avian infectious bronchitis.

The present invention relates, in general, to the genetics of viruses. In particular, the present invention provides methods of distinguishing between serotypes of avian infectious bronchitis virus based on restriction fragment length polymorphisms derived from the region of the S1 gene of IBV, peptides derived from the restriction fragments, related primers for polymerase chain reaction, and certain restriction length polymorphism patterns.

The invention provides a preparation method for a triple inactivated vaccine of recombinant Newcastle disease, bird flu and infectious bronchitis. The technical scheme is realized by gene modification of a Newcastle disease virus (DNV) LaSota strain. A recombinant NDV low virulent strain (rLaSota strain) is taken as a vector, firstly an S1 gene of a chicken infectious bronchitis virus (IBV) is inserted between F-HN, a recombinant NDV rLaSota/S1 expressing an S1 protein is constructed by utilizing a reverse genetic technology, and a result shows that rLaSota can serve as a live vector for stably expressing the S1 protein; secondly a recombinant NDV rLaSota/S1-HA expressing an HA gene is constructed, namely, the HA gene is inserted between P-M of the recombinant NDV rLaSota/S1; and thirdly immunogenicity assessment is performed. A result shows that the triple inactivated vaccine of the recombinant Newcastle disease, the bird flu and the infectious bronchitis, prepared through a conventional inactivated vaccine process has relatively good protection effects for the Newcastle disease, the bird flu and the infectious bronchitis.

The invention relates to an avian infectious bronchitis virus quick detection kit based on loop-mediated isothermal amplification technology and an application method thereof. The kit is composed of a (1) reaction solution, an (2) enzyme solution, a (3) primer solution and a (4) positive control solution, wherein the primer solution comprises three pairs of specific primers, i.e. inner primers, outer primers and loop primers. Before the reaction, a calcein-manganese complex is previously added, manganese is bonded with pyrophosphate ions precipitated by dNTP (deoxyribonucleotide triphosphate)to release calcein, and the result can be visually inspected and identified, wherein greenish yellow indicates a positive result, and light yellow indicates a negative result. The invention solves the problems of long period, low sensitivity, difficulty in field application and the like in the detection of avian infectious bronchitis virus in the prior art.

The invention discloses an animal epidemic disease three-color fluorescence RT-PCR detection kit and a detection method thereof, and is characterized in that the kit comprises multiple fluorescence RT-PCR reaction mother liquor, a positive control, a negative control, an AMV reverse transcriptase, and a Taq polymerase, wherein the multiple fluorescence RT-PCR reaction mother liquor contains a multiple 10*fluorescence RT-PCR reaction buffer, an avian influenza primer and a probe, a newcastle disease virus primer and a probe, an avian infectious bronchitis primer and a probe, and bovine serum albumin, wherein sequences of the forward primers, reverse primers, and specific probes are as shown in SEQIDNO. 1, NO. 2, NO.3, NO.4, NO.5, NO.6, NO.7. NO.8, NO.9, and NO.10. The advantages of the invention are that avian influenza, newcastle disease, and avian infectious bronchitis viruses can be detected simultaneously in a same reaction tube; the specificity is strong; the sensitivity is high, is rapid and accurate.

The invention discloses a triple egg yolk antibody against newcastle disease virus, avian influenza virus and infectious bronchitis virus and a preparation method and application thereof. The preparation method of the egg yolk antibody comprises the following steps of: (1) selecting a hen; (2) performing triple inactivation immunization of the hen obtained by the step (1) by use of newcastle disease virus, infectious bronchitis virus and avian influenza virus; (3) taking the egg laid by the hen and separating the egg yolk liquid; and (4) separating and purifying an egg yolk antibody from the egg yolk liquid obtained by the step (3). The triple egg yolk antibody disclosed by the invention is of great value in treating and preventing newcastle disease, avian influenza and infectious bronchitis.

The invention relates to a primer used in RT-PCR detection of chicken infectious bronchitis virus, a kit comprising the primer and a detection method thereof. The primer includes a specific primer pair used for amplifying a 5-terminal non-coding region conserved sequence of chicken infectious bronchitis virus (IBV). The specific primer pair is composed of a primer 1 and a primer 2, wherein the primer 1 is a single-chain DNA represented in the Seq ID No.1, the Seq ID No.2 or the Seq ID No.3 in the sequence table, and the primer 2 is a single-chain DNA represented in the Seq ID No.4, the Seq ID No.5 or the Seq ID No.6 in the sequence table. Tests comprising specificity, sensitivity, stability and repeatability prove that the kit can only specifically amplify IBV nucleic acid and is excellent in linearity relationship within the range of 1*10<2>-1*10<7> copies/[mu]L. The sensitivity of the primer reaches 10<2> copies/[mu]L. The primer is suitable for quantitative detection of various tissue, cell, body fluid samples and vaccine production intermediate products.

The invention relates to a method for producing a quadruple inactivated vaccine for newcastle disease, infectious bronchitis, avian influenza (H9 subtype) and infectious bursal disease. In the method, the inactivated vaccine is prepared by adopting a newcastle disease virus La Sota strain, an infectious bronchitis virus M41 strain, an avian influenza virus (H9 subtype) YBF003 strain, and an Escherichia coli genetic engineering bacteria E. coil BL21/pET28a-VP2 strain which expresses an infectious bursal disease virus VP2 protein serving as production bacteria toxic species, a super concentration process and a high-quality adjuvant. After immunizing animals, antibodies are produced rapidly; the potency is high; the protection period is long; and the outbreak and the spreading of epidemic diseases are reduced.

The invention discloses a visual protein chip for detecting serum antibody of new-castle disease virus of chickens, infectious bronchitis virus of chickens, avian influenza virus and infectious bursal disease virus of chickens , which is prepared by the following steps: purifying and diluting whole proteins of the four virus respectively; pointing samples of the positive control serum, the negative control serum and the four virus proteins onto a chip carrier respectively; drying, fixing, sealing and washing the samples to obtain the visual protein chip. The visual protein chip uses the purified whole proteins as capturing antigens to detect the virus-specific antibodies in chicken serum so as to simplify the preparation technology and reduce the production cost, and the visual protein chip has better specificity but no cross, has high reliability of results and has the advantages of quickness, simplicity and convenience, high sensitivity, good specificity and the like. When the serum is diluted by 6,400 times, the visual protein chip still can detect the antibodies, the sensitivity is 400 times of that of the prior AGP detection method. According to the detection to serum samples in-place, the detection rate of the visual protein chip is higher than the proir AGP method remarkably.

The invention relates to a method for producing a newcastle disease, infectious bronchitis, egg drop syndrome and avian influenza (H9 subtype) combined inactivated vaccine. A newcastle disease virus La Sota strain, a gallinaceous infectious bronchitis virus M41 strain, an egg drop syndrome virus NE4 strain and an A-type avian influenza virus (H9 subtype) YBF003 strain serve as production virus strains, and are mixed with a high-quality adjuvant to be prepared into the inactivated vaccine through a super concentration process; after an animal is immunized by the vaccine, an antibody is rapidly produced, the titer is high, the protection period is long, and occurrence and spread of diseases are reduced.

The invention discloses a method for quickly constructing an IBV (Avian Infectious Bronchitis Virus) reverse genetic strain, and belongs to the technical field of coronavirus reverse genetics. The constructing method comprises the following steps: quickly completing construction containing IBV genomic full-strength cDNA (Complementary Deoxyribose Nucleic Acid) clone by taking a BAC (Bacterial Artificial Chromosome) vector as a framework and applying an in-vitro homologous recombination technology, directly transfecting cells by a constructed recombinant plasmid, and transcribing in the cells, thus obtaining a transcript having infectivity; completing virus packaging; inoculating SPF (Specific Pathogen Free) chick embryo to a mixed solution of the cells and a culture medium and passing from generation to generation, thus obtaining the IBV reverse genetic strain. The constructing method disclosed by the invention has the advantages of simple operation and high positive cloning efficiency, the obtained IBV reverse genetic strain has passage stability, and an effective tool is provided for researching pathogenesis of the virus in vitro, developing a novel vaccine and the like; according to the method disclosed by the invention, transcription is carried out in the cells by utilizing a CMV (Cytomegalovirus) promoter added on a 5' terminal, and the rescue efficiency of the virus is greatly increased by utilizing an HDVR (Hepatitis Delta Virus Ribozyme) sequence added on a 3' terminal.

The invention relates to a live vaccine for infectious chicken bronchitis. The live vaccine consists of an antigen and a vaccine adjuvant, wherein the antigen comprises infectious chicken bronchitis viral strain with a collection code CGMCC No. 6752. The prepared vaccine can be used to prevent glandular stomach-type infectious chicken bronchitis. Furthermore, the infectious chicken bronchitis virus constituting the vaccine is a low virulent strain obtained by passage several times, thus reducing the side effect caused by the existing infectious bronchitis virus as the antigen.

The invention provides a method for production of an infectious bronchitis virus from a cell line. The employed cell line can be a Vero cell line, a BHK-21 cell line, a PK-15 cell line, a Marc145 cell line, a ST cell line, and an IBRS-2 cell line. The method includes: when the cell line forms a well-grown cell monolayer, inoculating an infectious bronchitis virus and making it adsorbed to the cell line; using a cell maintenance medium to conduct culture, adding an incubation agent to incubate the cell for 20-40min, and performing infectious bronchitis virus proliferation; and when CPE reaches over 75%, harvesting the infectious bronchitis virus. The method for production of an infectious bronchitis virus vaccine from a cell line provided in the invention can achieve the purpose of more secure and more effective infectious bronchitis virus and vaccine production.

The invention relates to the technical field of biological detection, and aims at providing a kit for RT-PCR typing detection of an avian infectious bronchitis virus. The kit comprises the following three pairs of primers including the universal primers designed according to an S1 gene segment of the infectious bronchitis virus, the 4/91 type virulent strain specific primers and the kidney type virulent strain specific primers designed according to an nsp12 gene segment, and the sequences of the three pairs of the primers are shown in SEQ ID NO:6-11. According to the kit, the purpose of performing typing detection on the IBV can be achieved only through one time of an RT-PCR reaction, and identification detection on the IBV virulent strains of the kidney type, the 4/91 type and the respiration type through the one-time RT-PCR method is achieved for the first time in the industry; the advantages of being convenient and rapid to use, high in sensibility and specificity and the like are achieved, and a novel effective detection means is supplied to clinical rapid identification diagnosis of the IBV, epidemiological investigation and IBV prevention and control.

The invention discloses a method for constructing a coronavirus infectious clone and an application thereof. The method comprises the following steps: firstly, acquiring a cDNA segment covering a selected full-length genome of coronavirus; performing segmental cloning; adopting a RED/ET recombination technology for assembling the cDNA segment containing the full-length genome of coronavirus onto aplasmid vector; screening, thereby acquiring the infectious clone containing the full-length cDNA of coronavirus. The invention has the beneficial effects that a medium/low copy plasmid vector is firstly utilized to successfully construct the infectious bronchitis coronavirus infectious clone; a new method is established for the coronavirus infectious clone; the method is capable of solving the problems of difficulty in selecting coronavirus vector and instability of viral macro-genome in bacteria; the positive cloning efficiency is high; the acquired reverse genetic vaccine strain cloning has integrity, passage stability, infectivity and high rescuing efficiency; an effective tool is supplied for researching nosogenesis of coronavirus, developing a novel vaccine, and the like.

The invention discloses a dual reverse transcription-polymerase chain reaction (RT-PCR) detection method for identifying an H9N2 subtype avian influenza virus. A pair of primers is designed respectively according to HA genes and NA genes of the H9N2 subtype of avian influenza virus (AIV), the HA genes and NA genes of the H9N2 subtype AIV in a sample can be subjected to specific amplification, and lengths of target fragments are respectively 700bp (HA) and 423bp (NA). According to the method, cross reaction is avoided in subtype AIV such as H3N8, H4N6 and H5N8, Newcastle disease virus and infectious bronchitis virus of chicken; the lowest detection amount of allantoic fluid of the virus is 1*103.25EID50/100uL; compared with conventional methods such as hemagglutination inhibition of virus and a neuraminidase inhibition test, the method has the advantage that the coincidence rate of the identification result is 100 percent. A rapid, specific and sensitive detection means is provided for identifying the H9N2 subtype AIV. The detection method can be used for rapidly diagnosing diseases caused by the H9N2 subtype AIV and has good application prospects in aspects of clinical diagnosis and epidemiological investigation.

The invention discloses an LDL-T strain as an attenuated vaccine strain for avian infectious bronchitis and application thereof, which belong to the field of prevention and treatment of the avian infectious bronchitis. The attenuated live vaccine strain CGMCC No.12549 for the avian infectious bronchitis provided by the invention has good safety and immune protection, the immune protection rate reaches 80 percent or more, and the LDL-T strain has good protective efficacy on TW I type avian infectious bronchitis viruses, and can effectively protect chickens against the attack of the TW I type infectious bronchitis viruses with strong virulent; moreover, the LDL-T strain is safe for chickens and laying breeders, side reaction does not exist, and the safety is good. The cold-adapted attenuated vaccine strain disclosed by the invention can be applied to the preparation of single vaccines, mixed vaccines and the like for preventing and treating the avian infectious bronchitis viruses.

The invention relates to an artificially weakened infectious bronchitis virus and application thereof. The artificially weakened infectious bronchitis virus uses the infectious bronchitis virus (IBV) which has good immunogenicity and is separated by an inventor to be passaged and attenuated, so as to obtain an attenuated strain FNO-E55 as a virus seed for production to prepare a safe and effective infectious bronchitis live vaccine. The strain used in the invention can effectively prevent occurrence of infectious bronchitis resulted from the infectious bronchitis virus (793/B serotype) by aiming at a prevalence situation of the current domestic infectious bronchitis.

The invention discloses an application of avian infectious bronchitis virus vaccine strain in the preparation of inactivated vaccine. The separated wild virulent strain of infectious bronchitis virus is subjected to chick embryo adaptability domestication so as to obtain an infectious bronchitis virus vaccine strain, which can well adapt to the chick embryo, maintains good hereditary characters and immunogenicity, and is named as ck/CH/LLN101135. Then the obtained infectious bronchitis virus vaccine strain is inactivated and subjected to immune efficacy evaluation experiments. The experiment results show that the infectious bronchitis inactivated vaccine prepared from the vaccine strain can trigger the organism to generate a good immune response, can prominently improve the motive immune protective effectiveness of chicken at the same time, and effectively protects chicken from being affected by infectious bronchitis virus. The infectious bronchitis virus vaccine strain can be used to prepare single vaccine or combined vaccine of avian infectious bronchitis.

The invention discloses an S1 and N double-antigen protein recombinant plasmid capable of expressing IBV (Infectious Bronchitis Virus) and a construction method and an application thereof. The inventor designs primers according to S1, N and Ub gene sequences of infectious bronchitis virus registered in GenBank, amplifies the S1 and N genes of China isolate GX-YL5 strain of the infectious bronchitis virus and a chicken Ub gene through PCR (Polymerase Chain Reaction), inserts the N and S1 genes of the China isolate GX-YL5 strain of the infectious bronchitis virus and the chicken Ub gene into an eukaryotic expression vector pVAX1 through a molecular biological method, thus constructing an expression plasmid capable of co-expressing the N and S1 genes and the chicken Ub gene and researches DNA vaccine Pvax1-Ub-linker-N-S1. Experiments prove that the vaccine has a significant curative effect of preventing IBV. The S1 and N double-antigen protein recombinant plasmid disclosed by the invention can be used for simultaneously inserting the S1 and N genes of IBV and the chicken Ub gene which are connected in series into the pVAX1 vector to prevent the infectious bronchitis virus.

An infectious bronchitis virus low virulent strain, a constructing method thereof and applications of the strain are disclosed. An infectious bronchitis virus high virulent strain of the QX-Like genotype, which is separated in the field, is adopted as a female parent, and is subjected to 100 or above generations of continuous passage through chicken embryos to obtain the attenuated virulent strain. The microbial preservation accession number of the low virulent strain is CGMCC No.13292. The S1 sequence of genome of the low virulent strain is shown as SEQ ID No.1. Chickens immunized with the low virulent strain do not die, and are free of obvious tissue organ lesions and clinical symptoms, thus proving that the low virulent strain is safe to chickens and good in safety. The challenge protection rate of the low virulent strain for immunized chickens is 80-100% so that the low virulent strain has good immunogenicity. Virulence enhancement tests prove that virulence of the low virulent strain is not enhanced, and the low virulent strain is stable in heredity and is suitable as a vaccine strain or a virus seed for preventing and treating infectious bronchitis.

The invention discloses a multi-fluorescent immunoassay method for rapidly distinguishing 6 types of poultry respiratory pathogens. The multi-fluorescent immunoassay method is simple to operate; a target amplified fragment is obtained through a PCR (Polymerase Chain Reaction); then an amplified product, fluorescence coded microspheres and streptavidin-phycoerythrin are hybridized; an MFI (Mean Fluorescence Intensity) value is read through a detector to distinguish viruses of different types. According to the method disclosed by the invention, avian influenza viruses, chicken infectious bronchitis viruses, chicken Newcastle disease viruses, chicken infectious laryngotracheitis viruses, mycoplasma gallisepticum and mycoplasma synoviae can be accurately detected at the same time; the multi-fluorescent immunoassay method has high specificity, high sensitivity and good repeatability. Compared with a traditional detection method, the method disclosed by the invention realizes simultaneous detection of a plurality of types of different target molecules in the same sample; the use amount of the sample is less; the method is simple and rapid to operate and the detection cost can be greatly reduced.

The invention provides an indirect ELISA (enzyme-linked immuno-sorbent assay) kit for detecting a nephropathogenic avian infectious bronchitis virus and an antibody thereof, and belongs to the technical field of biology. The indirect ELISA kit for detecting the nephropathogenic avian infectious bronchitis virus and the antibody thereof comprises an ELISA plate which is coated by DS10 monoclonal antibodies resisting the nephropathogenic avian infectious bronchitis virus and is combined with DS10 inactivated purified antigens resisting the nephropathogenic avian infectious bronchitis virus. The indirect kit for detecting the nephropathogenic avian infectious bronchitis virus and the antibody thereof has the advantages of specificity, sensitivity, quickness and simplicity and convenience in operation, and can be used for quickly diagnosing whether chickens are infected by the nephropathogenic avian infectious bronchitis virus or not and monitoring the immune antibody in actual production.

The invention provides an RT-RAA detection method for avian infectious bronchitis virus. The RT-RAA detection method comprises a primer pair for detecting the avian infectious bronchitis virus and a probe set. In the primer pair, the sequence of the forward primer is SEQ ID NO:1, the sequence of the reverse primer is SEQ ID NO:2, and the sequence of the probe is SEQ ID NO:3. The invention provides a method for rapidly detecting the avian infectious bronchitis virus based on molecular biology, so as to realize safe, specific, rapid, sensitive, simple and high-throughput rapid detection of the avian infectious bronchitis virus, thereby making up for the shortcomings of the existing traditional detection technology. In addition, the method is very suitable for on-site rapid detection. Compared with a PCR method, the RT-RAA method performs reaction at a constant temperature, the operation is simple, the shackles of complicated instruments are eliminated, temperature change is not required, and the reaction time is greatly shortened.

The invention relates to an eosinophilic kidney type avian infectious bronchitis virus (IBV) vaccine strain, in particular to an eosinophilic kidney type IBV virulent vaccine strain and application thereof, belonging to the technical field of organisms. The eosinophilic kidney type avian IBV virulent vaccine strain has CGMCC (China General Microbiological Culture Collection) No. 3957, and fiber axon protein of the eosinophilic kidney type avian IBV virulent vaccine strain has a gene sequence shown as SEQ ID NO:1. The invention provides a vaccine composition which comprises the eosinophilic kidney type avian IBV virulent vaccine strain and pharmaceutically acceptable adjuvants or carriers. The eosinophilic kidney type IBV virulent vaccine strain provided by the invention has favorable immunogenicity to the current epidemic eosinophilic kidney type IBV. A vaccine prepared by a DS10 virus not only can effectively resist the attack of self virulent strains, but also can effectively prevent the attack of a respiratory vaccine strain, therefore, the avian IBV can be effectively prevented and treated.