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A kit for identifying strong and weak strains of chicken infectious bursal disease virus based on rt-pcr and rflp technology and its application

A technology for bursal disease and chicken infectivity, which is applied in the field of identifying strong and weak strains of chicken infectious bursal disease virus, and can solve the problems of lack of sensitivity, specificity, and rapid distinction of super-strong strains

Active Publication Date: 2021-01-12
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ELISA test, virus neutralization test (VNT), real-time RT-PCR and agar gel precipitation test (AGPT) have been used to detect and distinguish various IBDV strains, but there is still a lack of sensitivity, specificity and rapid differentiation of super-virulent strains and attenuated strains, especially clinical assays to differentiate infection from immunity

Method used

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  • A kit for identifying strong and weak strains of chicken infectious bursal disease virus based on rt-pcr and rflp technology and its application
  • A kit for identifying strong and weak strains of chicken infectious bursal disease virus based on rt-pcr and rflp technology and its application
  • A kit for identifying strong and weak strains of chicken infectious bursal disease virus based on rt-pcr and rflp technology and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Embodiment 1 The establishment of RT-PCR combined with RFLP detection IBDV method

[0086] 1. Method

[0087] 1.1 Sequence analysis and selection of enzyme cutting sites

[0088] A total of 14 IBDV strains with known virulence were selected for sequence analysis. For HZ-2 and JD-1 strains, the nucleotide sequences of the selected strains have been extensively studied and included in the NCBI database. The basic information of the strains is shown in Table 1. Comparing the nucleotide sequences of the above 14 strains by DNAMAN software, it is determined that the difference between the IBDV super-virulent strain and the attenuated strain is mainly concentrated in the important structural protein VP2 gene region of IBDV, and the sequence of the strong and weak strains is analyzed by NEBcutter software The specific enzyme cutting site of the base mutation site in the region where the differences are concentrated, finally selected three kinds of restriction endonucleases, Sp...

Embodiment 2

[0141] Embodiment 2 animal regression experiment identification IBDV strain virulence

[0142] 1. Method

[0143] 1.1 Rejuvenation of the virus

[0144] Take out 15 IBDV strains CEF94, BD, CA, HR, CF, SD, BC, DB11, DN-04, C-8, 05-6, 99-3, JC preserved in our laboratory from the -80°C refrigerator -7, SD-F3 and GC-7, centrifuge at 7000rpm 4°C for 10min, draw the supernatant with a disposable syringe, dilute the virus with sterilized PBS 100, and inoculate the diluent chorioallantoic membrane into 10-day-old SPF chicken embryos, Two chicken embryos were inoculated with the virus dilution. Each chicken embryo was inoculated with about 200uL virus liquid. The inoculated chicken embryos were sealed with paraffin, and then placed in a 37°C incubator for incubation. The chicken embryos that died within 24 hours were discarded, and the embryos were inspected twice a day. The dead chicken embryos were taken out at any time until 96 hours. All the chicken embryos were taken out toge...

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Abstract

The invention discloses a kit for distinguishing high virulent strain of infectious bursal disease virus from low virulent strain of infectious bursal disease virus on basis of RT-PCR and RFLP technologies and application thereof. The kit comprises a primer pair used for amplifyin infectious bursal disease virus gene and SpeI, SacI and StuI restriction enzyme. The identification and diagnosis method based on the RT-PCR and RFLP technologies can distinguish very virulent virus strain of IBDV from low virulent strain, is simple in step and convenient to operate, and a novel effective technical means is provided for the rapid identification and diagnosis of high virulent strain and low virulent strain of the IBDV.

Description

technical field [0001] The invention relates to a kit for distinguishing strong and weak strains of chicken infectious bursal disease virus and its application, in particular to a kit for distinguishing strong and weak strains of chicken infectious bursal disease virus based on RT-PCR and RFLP technology Kits and their applications. The invention belongs to the technical field of virus detection. Background technique [0002] Chicken infectious bursal disease (Infectious bursal disease, IBD) is caused by infectious bursal disease virus (Infectious bursal disease virus, IBDV) acute, highly contact, lymphocytic infectious disease of chickens. IBD mainly infects chickens aged 3 to 6 weeks, and mainly invades the central immune organ, the bursa of Fabricius, leading to immunosuppression. There are two distinct types of IBDV, called serotype I and serotype II. Serum type I virus is pathogenic to chickens; serum type II is isolated from turkeys and has no pathogenicity to chick...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12Q1/683C12R1/93
CPCC12Q1/683C12Q1/686C12Q1/701C12Q2521/107C12Q2525/131
Inventor 唐丽杰周晗王丽徐义刚李一经韩冰林庆宇
Owner NORTHEAST AGRICULTURAL UNIVERSITY