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Indirect ELISA kit for detecting novel duck reovirus infection antibody

A virus infection and duck reovirus technology, applied in the direction of virus/bacteriophage, virus, virus peptide, etc., can solve the problems of inability to distinguish new duck reovirus antibodies, unfavorable virus purification, etc., and achieve low cost, rapid detection and repeatability good sex effect

Pending Publication Date: 2021-03-12
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it has been reported that the ELISA method for detecting NDRV serum antibodies cannot distinguish whether the new duck reovirus antibodies are from inactivated vaccine immunization or virus infection, which is not conducive to the purification of the virus in farms

Method used

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  • Indirect ELISA kit for detecting novel duck reovirus infection antibody
  • Indirect ELISA kit for detecting novel duck reovirus infection antibody
  • Indirect ELISA kit for detecting novel duck reovirus infection antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1, the preparation of novel duck reovirus p18 recombinant protein

[0020] (1) Cloning of p18 protein gene

[0021] The protein sequence of the non-structural protein p18 of the novel duck reovirus is shown as SEQ ID NO: 2. Using the genomic DNA of the new duck reovirus NDRV SY strain (GenBankMK955818to MK955827.) as a template, and using P18-F and P18-R as primers, the gene of the nonstructural protein p18 protein was amplified by high-fidelity enzyme PCR, and the p18 Restriction sites Bam HI and Hind III were introduced into the two ends of the protein gene respectively.

[0022] The sequence of P18-F is as follows: 5'-CCA GGA TCC ATG TCA CTC CCG CCA ACC-3' (the italic part is the BamH I restriction site); the sequence of P18-R is as follows: 5'-CCA AAG CTT G GCA GTT GTT GAT TGT AGA T-3' (the part in italics is the Hind III restriction site).

[0023] The amplified target gene (SEQ ID NO: 1) was cloned into the vector pEASY-Blunt (Beijing Quanshijin Biot...

Embodiment 2

[0028] Embodiment 2, preparation of negative and positive control serum

[0029] 1. Positive control serum for new duck reovirus infection: take 60 healthy non-immune ducks after 3 to 5 days of age, and adopt ducks of new duck reovirus NDRV-SY strain (GenBank accession number: MK955818-MK955827) Embryo tissue toxicity is 1000ELD per duck 50 Inoculation, blood collection and separation of serum every week, a total of 3 collections, the serum of each duck was combined and mixed, and the positive NDRV antibody was detected by indirect immunofluorescence test, which was used as the positive control serum of new duck reovirus infection. Both the positive sera after wild infection of new duck reovirus and the positive control serum of new duck reovirus infection contained NDRV antibody (P18 antibody).

[0030]The specific method of indirect immunofluorescence test detection is as follows: inoculate 100 PFU of NDRV-SY strain in each well of a 24-well cell culture plate, culture for ...

Embodiment 3

[0034] Embodiment 3, novel duck reovirus infection antibody indirect ELISA kit and detection method

[0035] 1. The composition of the new duck reovirus infection antibody indirect ELISA kit

[0036] New duck reovirus infection antibody indirect ELISA kit includes: washing solution, diluent, enzyme-labeled plate coated with p18 recombinant protein, enzyme-labeled secondary antibody, new duck reovirus infection positive control serum and negative control serum , chromogenic solution and stop solution.

[0037] (1) washing liquid

[0038] Washing solution (PBST buffer) is PBS buffer containing 0.05% (volume percent concentration) Tween-20. Prepare as follows: add Tween-20 with a final concentration (volume percentage concentration) of 0.05% and sodium azide with a final concentration of 5g / L in PBS buffer solution with a concentration of 0.01M and a pH value of 7.4.

[0039] The preparation method of PBS buffer solution with a concentration of 0.01M and a pH value of 7.4 is a...

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Abstract

The invention provides an indirect ELISA kit for detecting a novel duck reovirus infection antibody, and belongs to an immunological detection method in the field of biotechnology. The sequence of thenovel duck reovirus non-structural protein p18 is shown as SEQ ID No: 2. According to the indirect ELISA kit for detecting the novel duck reovirus infection antibody, whether the antibody is derivedfrom virus infection or inactivated vaccine immunity can be distinguished, so that the novel duck reovirus infection poultry group can be removed, and the transmission of the novel reovirus can be effectively controlled.

Description

technical field [0001] The invention belongs to an immunological detection method in the field of biotechnology, and in particular relates to an indirect ELISA kit for detecting antibodies infected by a new type of duck reovirus. Background technique [0002] Duck reovirus disease is caused by duck reovirus (Duck reovirus, DRV), a kind of waterfowl such as muscovy duck, semi-muscle duck, meat duck (including Peking duck, Cherry Valley meat duck), shelduck and goose. Acute infectious diseases can be divided into two genotypes of Muscovy duck reovirus (MDRV) and novel duck reovirus (NDRV). NDRV can infect a variety of waterfowl such as muscovy ducks, semi-muscular ducks, meat ducks and geese, causing "hemorrhagic necrotizing hepatitis" in muscovy ducks (also known as "new liver disease" in muscovy ducks) and "splenic necrosis" in meat ducks. The mortality rate caused by natural infection of NDRV is low, and the diseased ducks become stiff ducks after tolerance; the morbidity ...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543C12N15/46C12N15/70C07K14/14
CPCG01N33/56983G01N33/54306C07K14/005C12N15/70C12N2720/12022G01N2333/14G01N2469/20
Inventor 潘群兴张小飞卢凤英孙华伟赵莎
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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