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A kind of indirect ELISA detection kit and application for detecting novel chicken reovirus antibody

A detection kit, a technology for reovirus, 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, etc.

Active Publication Date: 2021-02-23
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Studies have confirmed that the pathogen of this high-incidence chicken infectious disease with arthritis as the main symptom is a new type of chicken reovirus, which is a new infectious disease. The biological characteristics and pathogenic mechanism of the virus Research is still in its infancy, and there is still a lack of effective vaccine immunization and preventive control measures, and no serological detection method for this new virus has yet been reported

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  • A kind of indirect ELISA detection kit and application for detecting novel chicken reovirus antibody
  • A kind of indirect ELISA detection kit and application for detecting novel chicken reovirus antibody

Examples

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preparation example Construction

[0042] (1) Preparation of coated antigen:

[0043] The whole gene sequence of the new chicken reovirus was obtained by next-generation sequencing technology. The sequences of the 10 gene fragments L1, L2, L3, M1, M2, M3, S1, S2, S3, and S4 in the whole genome sequence are shown in SEQ Shown in ID NO.3-SEQ ID NO.12; According to the σc protein gene sequence of the novel chicken reovirus obtained, design two primers:

[0044] Upstream primer σc-F: 5'-ATGGACGGATTAACTCAACAGCA-3'

[0045] Downstream primer σC-R: 5'-TTAGGTATCGATGCCCGTACGCAC-3'.

[0046] The cDNA obtained by reverse transcription was amplified with a length of 981bp σC gene sequence (shown in SEQ ID NO.13) by ordinary PCR method with the above primers, and purified by gel electrophoresis and gel recovery, and the purified The cDNA was cloned into the prokaryotic expression vector PET-32a(+) to construct the recombinant prokaryotic expression vector PET32a-σC; the recombinant prokaryotic expression vector PET-σC was...

Embodiment 1

[0070] Example 1: Preparation of Coated Antigen

[0071] 1.1 Design and synthesis of specific primers: A pair of primers were designed according to the σc protein coding region of the whole gene sequence of the novel chicken reovirus (preservation number: CCTCC NO: V201817), and the two primers were respectively equipped with BamHI and SalⅠ restriction sites , it is estimated that the 981bp gene fragment (shown in SEQ ID NO.13) in the novel chicken reovirus genome can be amplified.

[0072] Upstream primer σc-F: 5'-ATGGACGGATTAACTCAACAGCA-3';

[0073] Downstream primer σC-R: 5'-TTAGGTATCGATGCCCGTACGCAC-3'.

[0074] 1.2 Construction of PET32a-σC prokaryotic expression vector:

[0075] The σC 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 the positive colonies were picked and shaken to extract the T-σC recombinant pla...

Embodiment 2

[0083] Embodiment 2: Indirect ELISA detection novel chicken reovirus antibody

[0084] The purified recombinant protein σC in Example 1 was used as the coating antigen to establish an indirect ELISA method, and the square array method was used to explore the optimal protein coating concentration and serum dilution concentration for ELISA, as well as the optimal reaction conditions for ELISA. The final conditions are as follows:

[0085] 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. The detection well σC protein coating amount is 500ng / well;

[0086] 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;

[0087] 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 novel chicken reovirus antibody. The preparation method of the recombinant antigen of the orphan virus σC protein is as follows: using the whole gene sequence of chicken reovirus with the preservation number CCTCC NO: V201817 as a template, amplify the σC gene fragment by using a primer pair, construct a recombinant expression vector, and express it through prokaryotic The system expressed chicken reovirus σC protein recombinant antigen. The indirect ELISA detection kit of the present invention can quickly and effectively detect the new-type chicken reovirus antibody whose main symptoms are joint swelling and lameness, so as to monitor the epidemic situation of the new-type chicken reovirus in chicken flocks .

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 chicken reovirus antibody and its application. Background technique [0002] Avian orthoreovirus (ARV) belongs to the family Reoviridae and the genus Orthoreovirus. It is a pathogen that causes viral arthritis in chickens. It mainly infects broiler chickens, and chickens with both meat and eggs are also infected. ARV mainly invades the hock joints, toe joints, tibiometatarsal joints and tendons of chickens, causing joint synovitis or tenosynovitis. Diseased chickens usually show symptoms of lameness, squatting, reluctance to walk, loss of appetite or even extinction, resulting in low feed conversion rate, increased number of dead chickens, reduced production efficiency, and caused serious economic losses to the poultry industry. Since the disease was first reported in my country in the mid-1980s, the infection of AR...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/569
CPCG01N33/56983G01N33/6893
Inventor 刁有祥唐熠刘赫
Owner SHANDONG AGRICULTURAL UNIVERSITY
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