Indirect ELISA assay kit for detecting novel duck reovirus antibody and application of indirect ELISA assay kit

A detection kit, a technology for reovirus, which is applied in measurement devices, instruments, scientific instruments, etc., can solve the economic loss of breeding duck breeding industry, the reduction of qualified rate of meat ducks for slaughter, and the new virus serological detection method has not been reported. And other issues

Active Publication Date: 2018-10-19
SHANDONG AGRICULTURAL UNIVERSITY
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Problems solved by technology

The disease mainly leads to a decrease in the egg production of breeding ducks and a decrease in the weight of meat ducks, an increase in the feed-to-meat ratio, and a significant reduction in the qualified rate of meat ducks for slaughter, causing serious economic losses to the meat ducks and breeding duck breeding industry.
[0004] Studies have confirmed that the pathogen of this high-incidence duck infectious disease with arthritis as the main symptom is a new type of duck 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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  • Indirect ELISA assay kit for detecting novel duck reovirus antibody and application of indirect ELISA assay kit

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preparation example Construction

[0041] (1) Preparation of coated antigen:

[0042]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:

[0043] Upstream primer σc-F: 5'-ATGGACGGATTAACTCAACAGCAGCGA-3'

[0044] Downstream primer σC-R: 5'-ACCTAAGGTGTGGCGCCGTACGGG-3'.

[0045] Use the above primers to amplify the cDNA obtained by reverse transcription through the common PCR method to amplify the σC gene sequence (shown in SEQ ID NO.13) with a length of 1000 bp, and purify it by means of gel electrophoresis and gel recovery. The cDNA was cloned into the prokaryotic expression vector PET-28a(+) to construct the recombinant prokaryotic expression vector PET28a-σC; the recombinant prokaryotic expression...

Embodiment 1

[0069] Example 1: Preparation of Coated Antigen

[0070] 1.1 Design and synthesis of specific primers: A pair of primers were designed according to the whole gene sequence σc protein coding region of the new duck reovirus (preservation number: CCTCC NO: V201818), and the two primers were cut with BamH Ⅰ and Sal Ⅰ respectively site, it is estimated that a 1000bp gene fragment in the genome of the novel duck reovirus can be amplified.

[0071] Upstream primer σc-F: 5'-ATGGACGGATTAACTCAACAGCAGCGA-3';

[0072] Downstream primer σC-R: 5'-AGTCGACTTAGGTATCGATGCCCGT-3'.

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

[0074] 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 plasmid and the PET...

Embodiment 2

[0082] Embodiment 2: Indirect ELISA detection novel duck reovirus antibody

[0083] 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:

[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. The detection well σC protein coating amount is 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 serum to be tested...

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Abstract

The invention discloses an indirect ELISA assay kit for detecting a novel duck reovirus antibody. The kit contains an ELISA plate coated with a novel duck reovirus sigmaC protein recombinant antigen.A preparation method of the novel duck reovirus sigmaC protein recombinant antigen comprises the following steps: taking a complete genome sequence of the duck reovirus with the preservation number ofCCTCC NO:V201818 as a template; amplifying by using a primer pair to obtain sigmaC gene segments; constructing a recombinant expression vector; expressing a duck sigmaC protein recombinant antigen bya prokaryotic expression system. The indirect ELISA assay kit disclosed by the invention can quickly and effectively detect the novel duck reovirus antibody which is newly broken out and takes tarsaljoint swelling as a main symptom so as to monitor epidemic conditions of the novel duck reovirus in a duck plump.

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 duck reovirus antibody and its application. 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 2016, there has been a large-scale outbreak of infectious diseases in ground ducks in Shandong, Hebei, Henan and Jiangsu, with swelling of the tarsal joint as the main symptom. The disease mainly causes swelling of the tarsal joint of ducks of all ages, and the tarsal joint often contains a small amount of yellow Blood sample exudate; in severe cases, there is caseous exudate in the joint cavity, and the affected ducks have different degrees of lameness. The disease mainly leads to a decrease in egg production and w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/558
CPCG01N33/558G01N33/56983
Inventor 刁有祥唐熠王鸿志
Owner SHANDONG AGRICULTURAL UNIVERSITY
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