CN115825433A - ELISA detection kit for identifying goose circovirus and type 2 duck circovirus and application thereof - Google Patents

Abstract

The invention provides an ELISA detection kit for identifying goose circovirus and type 2 duck circovirus and application thereof. The ELISA detection kit comprises an ELISA plate A coated with goose circovirus ORF3 recombinant protein and an ELISA plate B coated with type 2 duck circovirus ORF3 recombinant protein. GoCV-ORF3-ELISA and DuCV-2-ORF3-ELISA serological detection methods are established by taking GoCV-ORF3 protein and DuCV-2-ORF3 protein as coating antigens. Can be used for carrying out serological epidemiological investigation of GoCV and DuCV-2 in duck group. The detection method of the invention also has the advantages of rapid and convenient detection, and the establishment of the invention can fill the blank of related fields at home and abroad.

Description

ELISA detection kit for identifying goose circovirus and type 2 duck circovirus and application thereof

Technical Field

The invention belongs to the field of aquatic bird infectious diseases, and particularly relates to an ELISA detection kit for identifying goose circovirus and type 2 duck circovirus and application thereof.

Background

According to The latest classification of The International Committee for viral classifications (The International Committee on Taxonomy of Viruses, ICTV), the circovirus family (Circoviridae) comprises two genera (Genus): circovirus (Circovirus) and Circovirus (Cyclovirus). Currently, the genus circovirus includes 49 members (specifices) of the Species psittacosis Beak-feather virus (BFDV), duck circovirus (Duck circovirus, duCV), goose circovirus (GoCV), pigeon circovirus (pivv), and porcine circovirus type 1, 2, 3, and 4 (PCV 1, PCV2, PCV3, PCV 4) ((specifices))https://talk.ictvonline.org/ taxonomy/)。

Enzyme linked immunosorbent assay (ELISA) is a qualitative and quantitative detection method in which soluble antigen or antibody is bound to a solid phase carrier such as polystyrene, and immunoreaction is carried out by utilizing the binding specificity of the antigen and the antibody. Common ELISA methods include indirect method, sandwich method, competitive method, etc.

The GoCV virus particles are non-enveloped, icosahedral-symmetric, single-stranded negative-strand circular closed DNA viruses with diameters of about 15 nanometers (nm). GoCV was discovered in 1999 by Soike et al from diseased geese with dysplasia and weight loss. The GoCV genome is 1821nt (or 1820 nt) in full length, has a classical neck-loop structure at its 5' -end, and is topped by 1 conserved 9 base sequence "TATTATT (1); the 3' -end intergenic region has a forward or reverse repeat sequence. Similar to the genome structure of other members of the circovirus genus, goCV also encodes two large Open Reading Frames (ORFs), the non-structural proteins (reps) associated with viral replication and the structural proteins (caps) associated with antigens located on the left side of the virus. In the GoCV genome, the third functional ORF newly identified on the complementary strand of ORF-V1, except the two major ORFs ORF-V1 and ORF-C1, is named ORF3, but the biological functions of the coding region and the virus biology of ORF3 gene deletion mutants have not been studied.

DuCV is an envelope-free, icosahedral symmetric virus whose genome is single negative strand circular DNA. Its genome encodes two large proteins (ORF-V1 and ORF-C1). ORF-V1 is located in the positive strand, encodes the replication associated protein (Rep) of DuCV, consists of 292 amino acids (aa), and is involved in viral replication; downstream of this, 1 TATA box may play a role in the regulation of Rep protein expression. ORF-C1 is distributed on the negative strand, encodes the DuCV nucleocapsid protein (Cap), consists of 257aa, the major antigenic region of the virus. In addition to the coding region, there are 2 intergenic regions associated with viral replication and proliferation: an intergenic region is located in the region between the 5' -ends of ORF-V1 and ORF-C1, and a hairpin structure is formed by 2 inverted repeats, a conserved nine-base (TATTATTAC) sequence is present in the stem loop, and two forward repeats of a six-base sequence (GTACTC) are also present in the vicinity of the stem loop region. Another highly variable region located in the intergenic region of the 3' -end of ORF-V1 and ORF-C1, contains 4 forward repeats (CACTTGGGCAG or CACTTGGCAG). The third functional ORF, newly identified in the DuCV-2 genome, located on the complementary strand of ORF-V1, in addition to the two major ORFs ORF-V1 and ORF-C1, is designated ORF3. In recent years, researches also prove that goose circovirus (GoCV) infection exists in duck groups, which brings practical requirements for a serological detection kit for goose circovirus (GoCV) and duck circovirus type 2 (DuCV-2).

Disclosure of Invention

The invention aims to provide an ELISA detection kit for identifying goose circovirus and duck circovirus type 2, application thereof and establishment of an ELISA detection method for identifying goose circovirus and duck circovirus type 2. The kit can be used for quickly and specifically identifying goose circovirus and type 2 duck circovirus.

The purpose of the invention is realized by the following technical scheme:

an ELISA detection kit for identifying goose circovirus and type 2 duck circovirus comprises an ELISA plate A coated with goose circovirus ORF3 recombinant protein and an ELISA plate B coated with type 2 duck circovirus ORF3 recombinant protein.

The preparation method of the goose circovirus ORF3 recombinant protein comprises the following steps:

after the ORF3 gene of goose circovirus is analyzed, the goose circovirus is inserted into a pET32a expression vector after the complete gene synthesis of related nucleotide sequences, the expressed goose circovirus ORF3 protein amino acid sequence is shown as SEQ ID NO.1, and a positive recombinant expression plasmid pET32a-GoCV-ORF3 is obtained after screening; transforming the recombinant expression plasmid into a prokaryotic expression competent cell, carrying out IPTG induced expression, and purifying an obtained expression product to obtain the goose circovirus ORF3 recombinant protein (GoCV-ORF 3 protein).

The preparation method of the 2-type duck circovirus ORF3 recombinant protein comprises the following steps:

after ORF3 gene of the duck circovirus type 2 is analyzed, the complete gene is synthesized into related nucleotide sequence, the nucleotide sequence is inserted into a pET32a expression vector to express the ORF3 protein amino acid sequence of the duck circovirus type 2 as shown in SEQ ID NO.2, and positive recombinant expression plasmid pET32a-DuCV-2-ORF3 is obtained after screening; and transforming the recombinant expression plasmid into a prokaryotic expression competent cell, carrying out IPTG induced expression, and purifying an obtained expression product to obtain the 2-type duck circovirus ORF3 recombinant protein (DuCV-2-ORF 3 protein).

The coating concentration of the goose circovirus ORF3 recombinant protein is 2.00 mu g/mL, and the coating concentration of the 2-type duck circovirus ORF3 recombinant protein is 2.00 mu g/mL.

The ELISA detection kit also comprises: coating solution, washing solution, sealant, sample diluent, enzyme-labeled secondary antibody, color developing agent, stop solution, negative control serum, goose circovirus positive control serum and type 2 duck circovirus positive control serum.

The coating solution is a carbonate buffer solution with 0.05M pH9.6, and the washing solution is a PBST buffer solution; the sealant is 5% of skimmed milk; the sample diluent is PBS buffer solution; the color developing agent is TMB. The stop solution is a 0.5M sulfuric acid solution; the enzyme-labeled secondary antibody is a goat anti-duck IgG enzyme-labeled secondary antibody.

The ELISA detection kit is applied to the identification of goose circovirus and type 2 duck circovirus.

An ELISA detection method for identifying goose circovirus and type 2 duck circovirus comprises the following steps:

(1) Coating: diluting a purified goose circovirus ORF3 recombinant protein (GoCV-ORF 3 protein) serving as a coating antigen with a carbonate buffer solution with the pH value of 0.05M and 9.6, adding the diluted solution into an ELISA plate A with 96 wells, coating the solution on each well by 100 mu L at 4 ℃ overnight; discarding redundant antigen in the coated wells, adding 200 mu L PBST into each well, washing for 4 times, and then patting the plate to be dry;

using purified duck circovirus type 2 ORF3 recombinant protein (DuCV-2-ORF 3 protein) as a coating antigen, diluting the antigen with 0.05M carbonate buffer solution with pH9.6, adding the diluted antigen into an ELISA plate B with 96 holes, wherein each hole is 100 mu L, and coating the antigen overnight at 4 ℃; discarding redundant antigens in the coated wells, adding 200 mu L of PBST into each well, washing for 4 times, and then patting the plate to be dry;

(2) And (3) sealing: respectively adding 200 mu L of 5% skim milk into each hole of the ELISA plate A and the ELISA plate B, sealing for 2h at 37 ℃; discarding redundant skim milk in the sealed hole, washing with PBST for 4 times, and drying;

(3) Adding serum to be detected: adding 100 mu L of serum diluted by PBS buffer solution into each hole of the ELISA plate A and the ELISA plate B respectively, and incubating for 1h at 37 ℃; discarding redundant serum in the incubated hole, washing with PBST for 4 times, and drying after washing;

(4) Adding a secondary antibody: adding 100 mu L of goat anti-duck IgG enzyme-labeled secondary antibody diluted by PBS buffer solution into each hole of the ELISA plate A and the ELISA plate B, and incubating for 1h at 37 ℃; then, discarding redundant enzyme-labeled secondary antibodies in the incubated holes, washing for 4 times by using PBST, and then patting dry after washing;

(5) Color development: adding 100 mu L of TMB color developing agent into each hole of the ELISA plate A and the ELISA plate B respectively, and developing for 7min in a dark place;

(6) And (4) terminating: 0.5M of H is added into each hole of the ELISA plate A and the ELISA plate B respectively ₂ SO ₄ Terminating the reaction; after the reaction is finished, placing the sample in a microplate reader, and reading the OD value of each sample at 450 nm;

(7) And (4) judging a result:

an indirect ELISA detection method established by the ELISA plate A, namely a GoCV-ORF3-ELISA method, is judged as GoCV positive if the OD value of serum to be detected is more than or equal to 0.506, and is judged as GoCV negative if the OD value is less than 0.506;

an indirect ELISA detection method established by the ELISA plate B, namely the DuCV-2-ORF3-ELISA method, is judged as DuCV-2 positive if the OD value of the serum to be detected is more than or equal to 0.481, and is judged as DuCV-2 negative if the OD value is less than 0.481;

the same serum is detected to be positive by a GoCV-ORF3-ELISA method and a DuCV-2-ORF3-ELISA method, and the serum is judged to be double positive to GoCV and DuCV-2;

the same serum is negative by the GoCV-ORF3-ELISA method and the DuCV-2-ORF3-ELISA method, and the serum is judged to be double negative by GoCV and DuCV-2;

the same serum is detected to be positive by a GoCV-ORF3-ELISA method, and is detected to be negative by a DuCV-2-ORF3-ELISA method, and the serum is judged to be GoCV positive and DuCV-2 negative;

the same serum is detected to be positive by a DuCV-2-ORF3-ELISA method, and is detected to be negative by a GoCV-ORF3-ELISA method, and the serum is judged to be positive by the DuCV-2 and negative by the GoCV;

goose circovirus and duck circovirus type 2 can be identified by combining the GoCV-ORF3-ELISA method and the DuCV-2-ORF3-ELISA method.

In the step (1), the antigen-coated goose circovirus ORF3 recombinant protein is diluted to 2.00 mu g/mL by using 0.05M carbonate buffer solution with pH9.6; the antigen-coated duck circovirus type 2 ORF3 recombinant protein is diluted to 2.00 mu g/mL by using a carbonate buffer solution with 0.05MpH9.6. Namely, the coating concentration of the goose circovirus ORF3 recombinant protein is 2.00 mu g/mL, and the coating concentration of the 2-type duck circovirus ORF3 recombinant protein is 2.00 mu g/mL.

In step (3), the serum was treated with PBS buffer at a ratio of 1: diluting by 80 times;

in the step (4), the goat anti-duck IgG enzyme-labeled secondary antibody is treated with PBS buffer solution according to the weight ratio of 1: and (4) diluting by 4000 times.

Compared with the prior art, the invention has the advantages that:

the research expresses ORF3 proteins of goose circovirus (GoCV) and duck circovirus type 2 (DuCV-2), and the purified proteins are used as antigens to establish serological detection methods and provide corresponding detection kits. The established detection method can be used for carrying out GoCV and DuCV-2 serological epidemiological investigation in duck groups.

The invention establishes GoCV-ORF3-ELISA and DuCV-2-ORF3-ELISA serological detection methods by taking GoCV-ORF3 protein and DuCV-2-ORF3 protein as coating antigens. Wherein, goCV-ORF3-ELISA established by the expressed GoCV ORF3 protein detects GoCV infected serum as positive; duCV-2-ORF3-ELISA established by the expressed ORF3 protein of DuCV-2 detects DuCV-2 infected serum as positive, and establishes a serological detection kit for identifying goose circovirus (GoCV) and duck circovirus type 2 (DuCV-2) after combination. The invention can fill up the blank of related research at home and abroad. The results of the detection of clinical samples show that the method can be applied to production practice as a method for detecting goose circovirus (GoCV) and duck circovirus type 2 (DuCV-2). The detection method of the invention also has the advantages of rapid and convenient detection.

Drawings

FIG. 1 is a SDS-PAGE picture of the GoCV-ORF3 protein; wherein, M: protein molecular weight standards; 1: is not induced; 2-3: induced protein of interest).

FIG. 2 is a Western Blot of the GoCV-ORF3 protein; wherein, M: protein molecular weight standards; 1: a protein of interest).

FIG. 3 is an SDS-PAGE picture of the DuCV-2-ORF3 protein; wherein, M: protein molecular weight standards; 1: is not induced; 2-4: induced protein of interest).

FIG. 4 is a Western Blot of the DuCV-2-ORF3 protein; wherein, M: protein molecular weight standards; 1: a protein of interest).

FIG. 5 is a diagram showing the specificity analysis of GoCV-ORF3-ELISA by the indirect ELISA method established with the purified GoCV-ORF3 protein.

FIG. 6 is a diagram showing the specificity of DuCV-2-ORF3-ELISA, which is an indirect ELISA method established with purified DuCV-2-ORF3 protein.

Detailed Description

The invention is described in detail below with reference to the drawings and examples:

example 1: preparation of goose circovirus ORF3 recombinant protein (GoCV-ORF 3 protein) and duck circovirus ORF3 recombinant protein type 2 (DuCV-2-ORF 3 protein):

prokaryotic expression of ORF3 protein of GoCV

1.1 sequence analysis

By theoretically evaluating the hydrophilicity and hydrophobicity, signal peptide, transmembrane domain, basic structure and the like of the ORF3 protein sequence of GoCV, according to the evaluation result, the plasmid is constructed in a whole gene synthesis mode and cloned to a pET32a expression vector, and a positive recombinant expression plasmid (pET 32a-GoCV-ORF 3) is obtained after screening, wherein the expressed GoCV-ORF3 protein amino acid sequence is as follows, wherein the underlined region is a tag sequence on the vector).

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAP KYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHM DSPDLGTDDDDKAMADIGSMPRRRDHWSDLFGLVHRLEGMSTLWNNILHYLRQIPELETARLFLPASLQAQQLSHSFADSGIPAGEVSLLYPLFLRQQSRTCNYPDCRTPQLHNNLQR

1.2 protein inducible expression

The recombinant expression plasmid is transformed into prokaryotic expression competent cell Rosetta (DE 3), and a single colony of the expression strain is picked up and cultured in a triangular flask filled with 12mL of LB liquid culture medium at 37 ℃ and 220rpm overnight. The overnight-cultured bacterial suspension was inoculated into 1L of LB medium at a ratio of 1. When the OD reached 0.6-0.8, IPTG was added to the medium at a final concentration of 0.2mM, 220rpm, and cultured at 37 ℃ for 4 hours. The cells were collected by centrifugation at 4000rpm for 10min and the supernatant was discarded. Dissolving the collected bacterial thallus by using a crushing Buffer, and ultrasonically crushing the thallus in ice bath with the power of 400W,20min, ultrasonic for 2s and pause for 6s as a cycle. After sonication, the cells were centrifuged at 12000rpm and 4 ℃ for 20min and subjected to SDS-PAGE analysis (the size of the target protein was approximately 29.28kDa, as indicated by the arrow in FIG. 1).

1.3 purification by Nickel Sepharose affinity chromatography

5mL of Ni-NTA was taken and the equilibration column was washed with Binding Buffer 5 times the bed volume at a flow rate of 5mL/min. After incubating the filler and the sample for 1h, the column was filled and the permeate was collected. The column was washed with 5 bed volumes of Binding Buffer at a flow rate of 5mL/min. Washing impurities with Wash Buffer at a flow rate of 2mL/min, and collecting the eluent. Eluting with Elution Buffer at the flow rate of 2mL/min, and collecting the eluate. The collected eluate was dialyzed against 50mM Tris,300mM NaCl,2mM DTT, pH 8.0. After dialysis, PEG20000 is concentrated, filtered by 0.45 μm filter membrane, and then packed in 1mL/tube, and frozen at-80 ℃. Western Blot analysis of the final purification of the GoCV-ORF3 protein is shown in FIG. 2 (the size of the protein of interest is approximately 29.28kDa, shown by the arrow in FIG. 2). The concentration of the purified GoCV-ORF3 protein is 0.30mg/ml.

Prokaryotic expression of DuCV-2-ORF3 protein

2.1 sequence analysis

Through theoretical evaluation of hydrophilicity and hydrophobicity, signal peptide, transmembrane domain, basic structure and the like of an ORF3 protein sequence of DuCV-2, according to an evaluation result, the invention adopts a whole-gene synthesis mode to construct a plasmid and clone the plasmid onto a pET32a expression vector, and a positive recombinant expression plasmid pET32a-DuCV-2-ORF3 is obtained after screening, wherein the amino acid sequence of the expressed DuCV-2-ORF3 protein is as follows, wherein the underlined region is a label sequence on the vector.

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAP KYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHM DSPDLGTDDDDKAMADIGSMSHRRAGRIDPYRLVHQLSGMSTLWSNILHYLRQIPGHERARLFLPATLQGRQLSHCFGDSGIPEGEVYRSRPFPLRRLRTCNCRACRTLKHEHNLQR

2.2 protein inducible expression

The recombinant expression plasmid is transformed into prokaryotic expression competent cell Rosetta (DE 3), and a single colony of the expression strain is picked up and cultured in a triangular flask filled with 12mL of LB liquid culture medium at 37 ℃ and 220rpm overnight. The overnight-cultured bacterial suspension was inoculated into 1L of LB medium at a ratio of 1. When the OD reached 0.6-0.8, IPTG was added to the medium at a final concentration of 0.2mM, 220rpm, and cultured at 37 ℃ for 4 hours. The cells were collected by centrifugation at 4000rpm for 10min and the supernatant was discarded. Dissolving the collected bacterial thallus by using a crushing Buffer, and carrying out ultrasonic crushing on the thallus in ice bath, wherein the power is 400W,20min, the ultrasonic treatment is carried out for 2s, and the suspension is 6s to form a cycle. After sonication, the cells were centrifuged at 12000rpm and 4 ℃ for 20min and subjected to SDS-PAGE analysis (the size of the target protein was about 29.23kDa, as indicated by the arrow in FIG. 3).

2.3 purification by Nickel Sepharose affinity chromatography

5mL of Ni-NTA was taken and the equilibration column was washed with Binding Buffer 5 times the bed volume at a flow rate of 5mL/min. After incubating the filler and the sample for 1h, the column was placed, and the permeate was collected. The column was washed with 5 bed volumes of Binding Buffer at a flow rate of 5mL/min. Washing impurities by a Wash Buffer at the flow rate of 2mL/min, and collecting the eluent. Eluting with Elution Buffer at the flow rate of 2mL/min, and collecting the eluate. The collected eluate was dialyzed against 50mM Tris,300mM NaCl,2mM DTT, pH 8.0. After dialysis, PEG20000 is concentrated, filtered by 0.45 μm filter membrane, and then packed in 1mL/tube, and frozen at-80 ℃. The final purification of the DuCV-2-ORF3 protein by Western Blot analysis is shown in FIG. 4 (the size of the protein of interest is approximately 29.23kDa, as indicated by the arrow in FIG. 4). The purified DuCV-2-ORF3 protein concentration was 0.40mg/ml.

Example two: establishment of indirect ELISA detection method

Establishment of ELISA method

(1) Coating: diluting a purified goose circovirus ORF3 recombinant protein (GoCV-ORF 3 protein) serving as a coating antigen with a carbonate buffer solution with the pH value of 0.05M and 9.6, adding the diluted solution into an ELISA plate A with 96 wells, coating the solution on each well by 100 mu L at 4 ℃ overnight; discarding redundant antigens in the coated wells, adding 200 mu L of PBST into each well, washing for 4 times, and then patting the plate to be dry;

using purified duck circovirus type 2 ORF3 recombinant protein (DuCV-2-ORF 3 protein) as a coating antigen, diluting the antigen with 0.05M carbonate buffer solution with pH9.6, adding the diluted antigen into an ELISA plate B with 96 holes, wherein each hole is 100 mu L, and coating the antigen overnight at 4 ℃; discarding redundant antigens in the coated wells, adding 200 mu L of PBST into each well, washing for 4 times, and then patting the plate to be dry;

(2) And (3) sealing: respectively adding 200 mu L of 5% skim milk into each hole of the ELISA plate A and the ELISA plate B, sealing for 2h at 37 ℃; discarding redundant skim milk in the sealed hole, washing with PBST for 4 times, and drying;

(3) Adding serum to be detected: and (3) adding 100 mu L of duck serum diluted by PBS buffer solution into each hole of the ELISA plate A and the ELISA plate B, and incubating for 1h at 37 ℃. Discarding redundant duck serum in the incubated hole, washing for 4 times by PBST, and drying after washing;

(4) Adding a secondary antibody: and adding 100 mu L of goat anti-duck IgG enzyme-labeled secondary antibody diluted by PBS buffer solution into each hole of the ELISA plate A and the ELISA plate B, and incubating for 1h at 37 ℃. Then, discarding redundant enzyme-labeled secondary antibodies in the incubated holes, washing for 4 times by using PBST, and then patting dry after washing;

(5) Color development: adding 100 μ L of TMB color developing agent into each hole of the ELISA plate A and the ELISA plate B, and developing for 7min in dark;

(6) And (4) terminating: 0.5M of H is added into each hole of the ELISA plate A and the ELISA plate B respectively ₂ SO ₄ The reaction was terminated. After the reaction was terminated, the reaction mixture was placed in a microplate reader, and the OD value of each sample was read at 450 nm.

(7) And (4) judging a result:

an indirect ELISA detection method established by the ELISA plate A, namely a GoCV-ORF3-ELISA method, is judged as GoCV positive if the OD value of serum to be detected is more than or equal to 0.506, and is judged as GoCV negative if the OD value is less than 0.506;

an indirect ELISA detection method established by the ELISA plate B, namely a DuCV-2-ORF3-ELISA method, is judged as DuCV-2 positive if the OD value of the serum to be detected is more than or equal to 0.481, and is judged as DuCV-2 negative if the OD value is less than 0.481;

the same serum is detected to be positive by a GoCV-ORF3-ELISA method and a DuCV-2-ORF3-ELISA method, and the serum is judged to be double positive to GoCV and DuCV-2;

the same serum is negative by the GoCV-ORF3-ELISA method and the DuCV-2-ORF3-ELISA method, and the serum is judged to be double negative by GoCV and DuCV-2;

the same serum is detected to be positive by a GoCV-ORF3-ELISA method, and is detected to be negative by a DuCV-2-ORF3-ELISA method, and the serum is judged to be GoCV positive and DuCV-2 negative;

the same serum is detected to be positive by a DuCV-2-ORF3-ELISA method, and is detected to be negative by a GoCV-ORF3-ELISA method, and the serum is judged to be positive by the DuCV-2 and negative by the GoCV;

goose circovirus and duck circovirus type 2 can be identified by combining the GoCV-ORF3-ELISA method and the DuCV-2-ORF3-ELISA method.

The method is used for optimizing the concentration of the coating antigen, the dilution of serum and the dilution of the enzyme-labeled secondary antibody, and the optimized conditions are as follows: the concentrations of the recombinant protein of the envelope antigen goose circovirus ORF3 and the recombinant protein of the type 2 duck circovirus ORF3 are respectively 2.00 mu g/mL and 2.00 mu g/mL, and the concentrations of the serum 1.

The OD values of 30 goose circovirus negative sera were measured by the above GoCV-ORF3-ELISA method, and the average value was determined

And Standard Deviation (SD), and the average value is added with 3 times of standard deviation to be used as a threshold value for judging positive

And (3) obtaining a threshold value of 0.506, namely judging that the GoCV antibody is positive if the OD value of the serum to be detected is more than or equal to 0.506, and judging that the serum is negative if the OD value is less than 0.506.

The OD values of 30 parts of DuCV-2-ORF3-ELISA were measured for DuCV-2 negative serum and the average value was determined

And Standard Deviation (SD), and the average value is added with 3 times of standard deviation to be used as a threshold value for judging positive

The threshold value is 0.481, namely DuCV-2 antibody is judged to be positive if the OD value of the serum to be detected is not less than 0.481, and the OD value is less than 0.481, and the result is judged to be negative.

Specificity analysis of ELISA method

4.1 specificity of GoCV-ORF3-ELISA

The OD values of AIV, ATmV, GPV, GHPyV, duCV-2 and GoCV positive sera were simultaneously determined by the GoCV-ORF3-ELISA method established by the above GoCV, and the results are shown in FIG. 5, wherein only the GoCV sera were positive in detection result, and the other pathogens were negative in detection result (see FIG. 5).

4.2DuCV-2-ORF3-ELISA specificity

The OD values of AIV, ATmV, GPV, GHPyV, duCV-2 and GoCV positive sera were simultaneously determined by the DuCV-2-ORF3-ELISA method established above, and the results are shown in FIG. 6, wherein only DuCV-2 test results are positive, and other pathogen test results are negative (see FIG. 6).

5 clinical applications

65 parts of duck serum samples which are clinically checked are detected by an established ELISA detection method, wherein 5 parts of GoCV-ORF3-ELISA positive samples are detected, and the GoCV infection positive rate is 7.69%; 19 parts of DuCV-2-ORF3-ELISA positive sample are detected, and the DuCV-2 positive rate is 29.23%; goCV-ORF3-ELISA and DuCV-2-ORF3-ELISA detected 2 positive samples, and the double positive rate of GoCV and DuCV-2 infection was 3.08%.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims (10)

1. An ELISA detection kit for identifying goose circovirus and type 2 duck circovirus, which is characterized in that: the enzyme label comprises an enzyme label plate A coated with goose circovirus ORF3 recombinant protein and an enzyme label plate B coated with type 2 duck circovirus ORF3 recombinant protein.

2. The ELISA detection kit of claim 1, wherein: the preparation method of the goose circovirus ORF3 recombinant protein comprises the following steps:

after the ORF3 gene of goose circovirus is analyzed, the goose circovirus is inserted into a pET32a expression vector after the complete gene synthesis of related nucleotide sequences, the expressed goose circovirus ORF3 protein amino acid sequence is shown as SEQ ID NO.1, and a positive recombinant expression plasmid pET32a-GoCV-ORF3 is obtained after screening; transforming the recombinant expression plasmid into prokaryotic expression competent cells, carrying out IPTG induced expression, and purifying the obtained expression product to obtain the goose circovirus ORF3 recombinant protein.

3. The ELISA detection kit of claim 1, wherein: the preparation method of the 2-type duck circovirus ORF3 recombinant protein comprises the following steps:

after ORF3 gene of the duck circovirus type 2 is analyzed, the complete gene is synthesized into related nucleotide sequence, the nucleotide sequence is inserted into pET32a expression vector to express the ORF3 protein amino acid sequence of the duck circovirus type 2 as shown in SEQ ID NO.2, and positive recombinant expression plasmid pET32a-DuCV-2-ORF3 is obtained after screening; transforming the recombinant expression plasmid into prokaryotic expression competent cells, carrying out IPTG induced expression, and purifying the obtained expression product to obtain the 2-type duck circovirus ORF3 recombinant protein.

4. The ELISA detection kit of claim 1, wherein: the coating concentration of the goose circovirus ORF3 recombinant protein is 2.00 mu g/mL, and the coating concentration of the 2-type duck circovirus ORF3 recombinant protein is 2.00 mu g/mL.

5. The ELISA detection kit of claim 1, wherein: it still includes: coating solution, washing solution, sealant, sample diluent, enzyme-labeled secondary antibody, color developing agent, stop solution, negative control serum, goose circovirus positive control serum and type 2 duck circovirus positive control serum.

6. The use of the ELISA detection kit of any one of claims 1-5 for the identification of goose circovirus and duck circovirus type 2.

7. An ELISA detection method for identifying goose circovirus and type 2 duck circovirus is characterized in that: the method comprises the following steps:

(1) Coating: diluting the purified goose circovirus ORF3 recombinant protein as a coating antigen with a carbonate buffer solution with the pH of 0.05MpH9.6, adding the diluted protein into an ELISA plate A with 96 holes, coating 100 mu L of the protein on each hole at 4 ℃ overnight; discarding redundant antigen in the coated wells, adding 200 mu L PBST into each well, washing for 4 times, and then patting the plate to be dry;

using purified 2-type duck circovirus ORF3 recombinant protein as a coating antigen, diluting the antigen with 0.05M carbonate buffer solution with pH9.6, adding the diluted antigen into an ELISA plate B with 96 holes, wherein each hole is 100 mu L, and coating the antigen overnight at 4 ℃; discarding redundant antigens in the coated wells, adding 200 mu L of PBST into each well, washing for 4 times, and then patting the plate to be dry;

(2) And (3) sealing: respectively adding 200 mu L of 5% skim milk into each hole of the ELISA plate A and the ELISA plate B, sealing for 2h at 37 ℃; discarding redundant skim milk in the sealed hole, washing with PBST for 4 times, and drying;

(3) Adding serum to be detected: adding 100 mu L of serum diluted by PBS buffer solution into each hole of the ELISA plate A and the ELISA plate B respectively, and incubating for 1h at 37 ℃; discarding redundant serum in the incubated hole, washing with PBST for 4 times, and drying after washing;

(4) Adding a secondary antibody: adding 100 mu L of goat anti-duck IgG enzyme-labeled secondary antibody diluted by PBS buffer solution into each hole of the ELISA plate A and the ELISA plate B, and incubating for 1h at 37 ℃; then, discarding redundant enzyme-labeled secondary antibodies in the incubated holes, washing for 4 times by using PBST, and then patting dry after washing;

(5) Color development: adding 100 mu L of TMB color developing agent into each hole of the ELISA plate A and the ELISA plate B respectively, and developing for 7min in a dark place;

(6) And (4) terminating: 0.5M of H is added into each hole of the ELISA plate A and the ELISA plate B respectively ₂ SO ₄ Terminating the reaction; after the reaction is finished, placing the sample in a microplate reader, and reading the OD value of each sample at 450 nm;

(7) And (4) judging a result: an indirect ELISA detection method established by an ELISA plate A coated with goose circovirus ORF3 recombinant protein, namely a GoCV-ORF3-ELISA method, is characterized in that if the OD value of the serum to be detected is more than or equal to 0.506, the serum is judged to be GoCV positive, and if the OD value is less than 0.506, the serum is judged to be GoCV negative;

an indirect ELISA detection method established by an ELISA plate B coated with 2-type duck circovirus ORF3 recombinant protein is a DuCV-2-ORF3-ELISA method, if the OD value of the serum to be detected is not less than 0.481, the serum is judged to be DuCV-2 positive, and if the OD value is less than 0.481, the serum is judged to be DuCV-2 negative;

the same serum is detected to be positive by a GoCV-ORF3-ELISA method and a DuCV-2-ORF3-ELISA method, and the serum is judged to be double positive to GoCV and DuCV-2;

the same serum is negative by the GoCV-ORF3-ELISA method and the DuCV-2-ORF3-ELISA method, and the serum is judged to be double negative by GoCV and DuCV-2;

the same serum is detected to be positive by a GoCV-ORF3-ELISA method, and is detected to be negative by a DuCV-2-ORF3-ELISA method, and the serum is judged to be GoCV positive and DuCV-2 negative;

the same serum is detected to be positive by a DuCV-2-ORF3-ELISA method, and is detected to be negative by a GoCV-ORF3-ELISA method, and the serum is judged to be positive by the DuCV-2 and negative by the GoCV;

goose circovirus and duck circovirus type 2 can be identified by combining the GoCV-ORF3-ELISA method and the DuCV-2-ORF3-ELISA method.

8. The ELISA detection method for identifying goose circovirus and duck circovirus type 2 according to claim 7, characterized in that: in the step (1), the antigen-coated goose circovirus ORF3 recombinant protein is diluted to 2.00 mu g/mL by using a carbonate buffer solution with 0.05MpH9.6; the antigen-coated duck circovirus type 2 ORF3 recombinant protein is diluted to 2.00 mu g/mL by using 0.05M carbonate buffer solution with pH9.6.

9. The ELISA detection method for identifying goose circovirus and duck circovirus type 2 according to claim 7, characterized in that: the preparation method of the goose circovirus ORF3 recombinant protein comprises the following steps:

after the ORF3 gene of goose circovirus is analyzed, the goose circovirus is inserted into a pET32a expression vector after the complete gene synthesis of related nucleotide sequences, the expressed goose circovirus ORF3 protein amino acid sequence is shown as SEQ ID NO.1, and a positive recombinant expression plasmid pET32a-GoCV-ORF3 is obtained after screening; transforming the recombinant expression plasmid into a prokaryotic expression competent cell, carrying out IPTG (isopropyl-beta-thiogalactoside) induction expression, and purifying an obtained expression product to obtain goose circovirus ORF3 recombinant protein (GoCV-ORF 3 protein);

the preparation method of the 2-type duck circovirus ORF3 recombinant protein comprises the following steps:

after ORF3 gene of the duck circovirus type 2 is analyzed, the complete gene is synthesized into related nucleotide sequence, the nucleotide sequence is inserted into pET32a expression vector to express the ORF3 protein amino acid sequence of the duck circovirus type 2 as shown in SEQ ID NO.2, and positive recombinant expression plasmid pET32a-DuCV-2-ORF3 is obtained after screening; and transforming the recombinant expression plasmid into a prokaryotic expression competent cell, carrying out IPTG induced expression, and purifying an obtained expression product to obtain the 2-type duck circovirus ORF3 recombinant protein (DuCV-2-ORF 3 protein).

10. The ELISA detection method for distinguishing goose circovirus from duck circovirus type 2 according to claim 7, characterized in that: in the step (3), the serum is diluted by PBS buffer solution according to a ratio of 1;

in the step (4), the goat anti-duck IgG enzyme-labeled secondary antibody is added with PBS buffer solution according to the proportion of 1: and (4) diluting by 4000 times.

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