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A kind of identification method of chicken infectious bronchitis virus

A chicken infectivity and identification method technology, which is applied in the identification field of chicken infectious bronchitis virus, can solve the problems of lack of objective judgment standards, time-consuming and other problems, and achieve the effect of fast detection speed and high accuracy of results

Inactive Publication Date: 2015-08-26
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0005] The present invention aims at the problems of time-consuming and lack of objective criteria for IBV isolation and identification in the prior art, and provides a fast and accurate IBV identification for the study of the characteristics of IBV virus that can interfere with chicken Newcastle disease virus (NDV) in chicken embryos Method, this method mainly adopts to inoculate the chicken embryo with the allantoic cavity of the sample to be tested, then inoculate the chicken Newcastle disease virus (Newcastle disease virus, NDV) attenuated strain LaSota, and finally identify whether IBV exists according to the detection result of the hemagglutination value of the allantoic fluid of the chicken embryo , because this method adopts the mode of indirect determination, compared with the existing technology, it has the characteristics of fast detection speed and no special cultivation, and can be widely used in laboratory diagnosis, antibody detection and IBV serum of chicken infectious bronchitis Type identification, in order to evaluate the immune effect of IB vaccine, monitor the epidemic situation, etc., to provide a theoretical basis for formulating scientific immunization procedures and effectively preventing and controlling the occurrence of IB

Method used

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  • A kind of identification method of chicken infectious bronchitis virus
  • A kind of identification method of chicken infectious bronchitis virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] 1.1 Sample collection and processing

[0019] The trachea, lung, kidney, fallopian tube, intestinal tract, liver, spleen and other diseased tissues of chickens that were clinically suspected to be infected with IBV were ground with a grinder, and then added with penicillin (2000IU / mL) and streptomycin (2mg / mL) ) in 0.01MPBS buffer solution (PH7.4), mix well, freeze and thaw repeatedly at -20°C for 3 times, centrifuge at 8000rpm and take the supernatant, inoculate at 4°C for 2h, then it can be used for chicken embryo inoculation.

[0020] 1.2 Inoculation of chicken embryos

[0021] The allantois of the above-mentioned treated samples was inoculated into five 9-day-old SPF chicken embryos, and the five SPF chicken embryos pre-inoculated with PBS buffer were used as controls. The two groups of chicken embryos were hatched at 37°C for 27 hours respectively, the dead chicken embryos were discarded, and 0.2ml of the living chicken embryos were inoculated into the allantoic c...

Embodiment 2

[0025] 2.1 IBV viral content (EID 50 ) Determination:

[0026] IBV standard vaccine strains H120, 491 and MA5 and isolates 002, 007, 008, 283, 369, 327, 356 (wherein, standard vaccine strain H120 was purchased from China Veterinary Drug Administration, 491 and MA5 were amplified from IBV live vaccine; The isolates were isolated in our laboratory and identified by RT-PCR sequencing.) After aseptic treatment, they were diluted 10 times with 0.01MPBS buffer solution respectively, and 10 -3 -10 -8 6 dilutions, each dilution was inoculated with 5 SPF chicken embryos aged 9-10 days, incubated at 37°C for 24 hours, discarded dead chicken embryos, and inoculated 0.2ml of surviving chicken embryos into the allantoic cavity (virus content: 200EID 50 ) NDV attenuated strain LaSota, hatched at 37°C, hatched to 48h to detect the HA titer of chicken embryo allantoic fluid. If the chicken embryo HA titer is 0-2log2, it is judged as IBV infection. Calculate the EID of each virus strain ac...

Embodiment 3

[0033] 3.1 Sample collection and processing

[0034] The trachea, lung, kidney, fallopian tube, intestinal tract, liver, spleen and other diseased tissues of chickens that were clinically suspected to be infected with IBV were ground with a grinder, and then added with penicillin (2000IU / mL) and streptomycin (2mg / mL) ) in 0.01MPBS buffer solution (PH7.4), mix well, freeze and thaw repeatedly at -20°C for 3 times, centrifuge at 8000rpm and take the supernatant, inoculate at 4°C for 4h, then it can be used for chicken embryo inoculation.

[0035] 3.2 Inoculation of chicken embryos

[0036] The allantois of the above-mentioned treated samples was inoculated into five 10-day-old SPF chicken embryos, and the five SPF chicken embryos pre-inoculated with PBS buffer were used as controls. The two groups of chicken embryos were hatched at 37°C for 30 hours respectively, the dead chicken embryos were discarded, and 0.2ml of surviving chicken embryos were inoculated into the allantoic c...

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Abstract

The invention relates to a method for identifying chicken infectious bronchitis virus (IBV). The method is to inoculate SPF chicken embryos into the allantoic cavity of the sample to be tested, and set the SPF chicken embryos inoculated with PBS buffer solution as a control at the same time. Chicken embryos were incubated at 37°C for 24-30 hours, and then inoculated with attenuated Newcastle disease virus strain at 200-2000 times the half infectious dose (EID50) of chicken embryos, and then incubated at 37°C for 48-72 hours to detect the hemagglutination value of chicken embryo allantoic fluid , according to the hemagglutination value judgment result. This method utilizes the characteristic that IBV virus can interfere with the proliferation of chicken Newcastle disease virus (NDV) in chicken embryos, and can be used for laboratory diagnosis, antibody detection and IBV serotype identification of chicken infectious bronchitis, so as to carry out chicken infectious bronchitis vaccine immunization The evaluation of the effect and the monitoring of the epidemic situation provide a theoretical basis for formulating a scientific immunization program and effectively preventing and controlling the occurrence of the disease.

Description

technical field [0001] The invention relates to a biotechnology, in particular to a method for identifying chicken infectious bronchitis virus. Background technique [0002] Chicken infectious bronchitis (Avian Infectious Bronchitis, IB) is an acute, highly contagious, viral disease caused by chicken infectious bronchitis virus (Infectious Bronchitis Virus, IBV). Chicken infectious bronchitis was first discovered in North Dakota, USA in 1930. It mainly harms the respiratory tract of 2-day to 3-week-old chicks. It is characterized by wheezing, mental fatigue, and a mortality rate of 40%-90%. According to the clinical symptoms caused by IBV infection in chickens, it can be divided into respiratory type, renal type, intestinal type and other disease types, as well as some variant intermediate types. With the widespread use of various IB vaccines and the common occurrence of clinical mixed infections, IBV-infected chickens increasingly lack typical clinical symptoms and patholo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70
Inventor 胡北侠张秀美杨少华许传田张琳黄艳艳任海松
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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