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Indirect ELISA detection kit for detecting novel goose astrovirus antibody and application thereof

A detection kit and astrovirus technology, applied in the field of biological products, can solve the problems of lack of vaccine immunization and prevention and control measures, unreported new virus serological detection methods, and economic loss of meat goose breeding industry.

Active Publication Date: 2018-12-07
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Severe urate deposition occurred in the body cavity and joints of the sick goose, which caused the gosling to grow slowly, increase the feed-to-meat ratio, and significantly reduce the pass rate of goose slaughter, causing serious damage to the goose breeding industry. Economic losses
[0004] Studies have confirmed that the pathogen of this high incidence goose infectious disease with gout as the main symptom is a new type of goose astrovirus, which is a new infectious disease. The biological characteristics and pathogenic mechanism of the virus are still unknown. In its infancy, effective vaccine immunization and preventive control measures are still lacking, and serological detection methods for this new virus have not yet been reported

Method used

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  • Indirect ELISA detection kit for detecting novel goose astrovirus antibody and application thereof

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preparation example Construction

[0041] (1) Preparation of coated antigen:

[0042] The whole gene sequence of the new goose astrovirus was obtained by next-generation sequencing technology, and its whole gene sequence is shown in SEQ ID NO.3; according to the obtained ORF1b protein gene of the new goose astrovirus, two primers were designed:

[0043]Upstream primer ORF1b-F: 5'-ATGTTTGAAAAATTTTTCTATC-3';

[0044] Downstream primer ORF1b-R: 5'-CCAGATTTGAGAAAAAGAAGTC-3'.

[0045] The ORF1b gene sequence (as shown in SEQID NO.4) with a length of 1287bp was amplified by the general RT-PCR method with the above primers, and purified by gel electrophoresis and gel recovery, and the purified product was cloned into prokaryotic expression In the vector PET-28a(+), the recombinant prokaryotic expression vector PET28a-ORF1b was constructed; the recombinant prokaryotic expression vector PET-ORF1b was transformed into BL21(DE3) competent cells, and the recombinant protein ORF1b was highly expressed by 1mM IPTG induction...

Embodiment 1

[0069] Example 1: Preparation of Coated Antigen

[0070] 1.1 Design and synthesis of specific primers: According to the whole gene sequence (shown in SEQ ID NO.3) of the new goose astrovirus (preservation number is CCTCC NO: V201808), a pair of primers were designed on the ORF1b protein gene sequence, and the two primers were The segments are respectively provided with BamHI and SalI restriction sites, and it is estimated that a 1287bp gene fragment (shown in SEQ ID NO.4) in the novel goose Astrovirus genome can be amplified.

[0071] Upstream primer ORF1b-F: 5'-ATGTTTGAAAAATTTTTCTATC-3';

[0072] Downstream primer ORF1b-R: 5'-CCAGATTTGAGAAAAAGAAGTC-3'.

[0073] 1.2 Construction of PET28a-ORF1b prokaryotic expression vector:

[0074] The ORF1b gene fragment was amplified by PCR using specific upstream and downstream primers, the purified fragment was ligated with the PMD18-T vector, the ligated product was transformed into DH5α competent cells, and positive colonies were pic...

Embodiment 2

[0082] Embodiment 2: Indirect ELISA detection novel goose Astrovirus antibody

[0083] The purified recombinant protein ORF1b in Example 1 was used as the coating antigen to establish an indirect ELISA method, and the optimal protein coating concentration and serum dilution concentration of ELISA, as well as the optimal reaction conditions of ELISA were explored by using the square array method. The final conditions are as follows:

[0084] 2.1 Coating: Dilute the antigen with 1×carbonate buffer (pH=9.6) at 1:1000, take 10 μL and coat it into a 96-well ELISA plate, wrap it in plastic wrap, incubate at 37°C for 2 hours, and use 0.05% Wash with Tween PBST three times, 4 min each time. Detection hole ORF1b protein coating amount 500ng / well;

[0085] 2.2 Sealing: Dilute 200 μL / well of 5% skimmed milk powder with PBST for sealing, incubate at 37°C for 1 hour, spin dry, wash with PBST 3 times, 4 minutes each time;

[0086] 2.3 Serum action conditions: Add 10 μL of the serum to be...

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Abstract

The invention discloses an indirect ELISA detection kit for detecting a novel goose astrovirus antibody. The kit includes an ELISA plate coated with a novel goose astrovirus ORF1b protein recombinantantigen. The preparation method of the novel goose astrovirus ORF1b protein recombinant antigen includes: taking the complete gene sequence of goose astrovirus with a preservation number of CCTCC NO:V201808 as the template, conducting primer pair amplification to obtain an ORF1b gene fragment, constructing a recombinant expression vector, and expressing the goose astrovirus ORF1b protein recombinant antigen by a prokaryotic expression system. The indirect ELISA detection kit provided by the invention can achieve rapid and effective detection of the new outbreak novel goose astrovirus antibodywith gout as the main symptom, thus monitoring the epidemic situation of novel goose astrovirus in a gaggle of geese.

Description

technical field [0001] The invention relates to the technical field of biological products, in particular to an indirect ELISA detection kit for detecting novel goose astrovirus antibody and its application. Background technique [0002] Astrovirus (AStV) is a kind of non-encapsulated, spherical, single-stranded positive-sense RNA virus with a diameter of 28-30nm. The genome is infectious and 6.4-7.9kb long. The infection of this virus has a certain prevalence. It can cause enteritis and diarrhea in humans or animals, sometimes accompanied by vomiting and abdominal pain. Astroviridae can be divided into mammalian Astrovirus genus and avian Astrovirus genus according to the different infected hosts. Avian Astrovirus can cause a variety of diseases in poultry. vary with each other. Goose astrovirus (GAstV), which mainly affects young goslings, has been prevalent in many provinces, increasing the mortality rate of geese, and greatly endangering the healthy development of goos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/56983
Inventor 刁有祥唐熠刘赫
Owner SHANDONG AGRICULTURAL UNIVERSITY
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