Indirect ELISA detection method of duck reovirus causing spleen necrosis

A technology of reovirus and duck reovirus, which is applied in the field of indirect ELISA detection of duck reovirus, can solve problems such as increased duck feed-to-meat ratio, economic loss of meat ducks, and lower pass rate of meat ducks for slaughter

Active Publication Date: 2018-12-11
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
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  • Claims
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Problems solved by technology

The feed-to-meat ratio of ducks increases, and the pass rate of meat ducks drops significantly, causing serious economic losses to the meat duck and duck breeding industry
[0004] Studies have confirmed that the pathogen of this high-incidence duck infectious disease with spleen necrosis as the main symptom is a new type of duck reovirus, which is a new infectious disease. 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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  • Indirect ELISA detection method of duck reovirus causing spleen necrosis

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preparation example Construction

[0042] (1) Preparation of coated antigen:

[0043]The whole gene sequence of the new duck 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 obtained σc protein gene sequence of the novel duck reovirus, two primers were designed:

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

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

[0046] The cDNA obtained by reverse transcription was amplified with a length of 980 bp 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-28a(+) to construct the recombinant prokaryotic expression vector PET28a-σC; the recombinant prokaryotic expression vector PET-σC was...

Embodiment 1

[0071] Example 1: Preparation of Coated Antigen

[0072] 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 new duck reovirus (preservation number: CCTCC NO: V201843), and the two primers were respectively equipped with BamHI and SalⅠ restriction sites , it is estimated that a 980bp gene fragment in the genome of the novel duck reovirus can be amplified.

[0073] Upstream primer σc-F: 5,-ATACGCCTGATACTTTCCCT-3';

[0074] Downstream primer σC-R: 5'-GCGCAATGAGAAGAGCTATTCATC-3'.

[0075] 1.2 Construction of PET28a-σC prokaryotic expression vector:

[0076] 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-σ recombinant plasmid. The T-σ recombinant plas...

Embodiment 2

[0084] Embodiment 2: Indirect ELISA detection novel duck reovirus antibody

[0085] 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:

[0086] 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;

[0087] 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;

[0088] 2.3 Serum action conditions: add 10 μL of the serum to be te...

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Abstract

The invention discloses an indirect ELISA detection kit of duck reovirus causing the spleen necrosis. The kit comprises an elisa plate coated with novel duck reovirus sigma C protein recombinant antigen; a preparation method of the novel duck reovirus sigma C protein recombinant antigen comprises the following steps: adopting a complete gene sequence of the duck reovirus with a preservation numberof CCTCC NO: V201843 as a template, performing the amplification by using a primer pair to obtain a sigma C gene fragment, establishing a recombinant expression vector, and expressing the duck reovirus sigma C protein recombinant antigen by virtue of a prokaryotic expression system. The indirect ELISA detection kit can rapidly and effectively detect the outbreak novel duck reovirus antibody withthe main symptom of hock swelling so as to monitor the prevalence state of the novel duck reovirus in ducks.

Description

technical field [0001] The invention relates to the technical field of biological products, in particular to an indirect ELISA detection method for duck reovirus that causes duck spleen necrosis. Background technique [0002] Avian reovirus belongs to the genus Orthoreovirus of the Reovirdae family and can cause a variety of diseases in poultry. Its clinical manifestations vary depending on the virus strain, virulence or infected host. . [0003] Since 2017, a large-scale outbreak of infectious diseases with spleen necrosis as the main symptom has occurred in land ducks in Shandong, Hebei, Henan, Jiangsu, Anhui and other places in my country. The disease mainly causes spleen enlargement, hemorrhage and necrosis in ducks of all ages, resulting in Egg-laying and meat ducks lost their appetite, became thin, and had poor growth and development. Several necrotic lesions, swelling, hemorrhage, and edge hyperplasia appeared in the spleen. The feed-to-meat ratio of ducks increases,...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/535G01N33/558
CPCG01N33/535G01N33/558G01N33/56983
Inventor 刁有祥唐熠王鸿志
Owner SHANDONG AGRICULTURAL UNIVERSITY
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