CN115856295A - ELISA detection kit for detecting deletion of ORF3 of duck circovirus type 1 and application thereof - Google Patents

Abstract

The invention provides an ELISA detection kit for detecting whether the ORF3 of the duck circovirus type 1 is deleted or not and application thereof, wherein the ELISA detection kit comprises an ELISA plate A coated with the Cap recombinant protein of the duck circovirus type 1 and an ELISA plate B coated with the ORF3 recombinant protein of the duck circovirus type 1. The invention establishes a DuCV-1 indirect ELISA detection method by taking Cap protein and ORF3 protein of the duck circovirus type 1 as envelope antigens, and can be used for researching whether ORF3 of the duck circovirus type 1 is deleted or not after combination. The detection method of the invention also has the advantages of rapid and convenient detection. The establishment of the invention can fill the blank of related fields at home and abroad.

Description

ELISA detection kit for detecting deletion of ORF3 of duck circovirus type 1 and application thereof

Technical Field

The invention belongs to the field of waterfowl infectious diseases, and particularly relates to an ELISA detection kit for detecting whether ORF3 of type-1 duck circovirus is deleted or not 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/)。

DuCV was first discovered in German Muscovy ducks by Hattermann et al in 2003. 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 DuCV replication associated protein (Rep), and consists of 292 amino acids (aa). ORF-C1 is distributed on the minus strand, encodes the DuCV nucleocapsid protein (Cap), and consists of 257 aa. There are 2 intergenic regions associated with viral replication and proliferation outside the coding region: 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 (TATTATTATTAC) 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 comprises 4 forward repeats (CACTTGGGCAG or CACTTGGCAG).

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, competition method, etc.; of these, indirect ELISA methods are most commonly used, mainly for the detection of serum antibodies. In the DuCV-1 genome, a newly identified third functional ORF located on the complementary strand of ORF-V1, except for the two major ORFs ORF-V1 and ORF-C1, was designated ORF3, but the viral biology and the like of ORF3 gene deletion mutants have not been studied. In order to establish a method for researching the biological functions of the ORF3 protein of DuCV-1, the Cap protein and the ORF3 protein of DuCV-1 are expressed respectively, and the combination of the Cap protein and the ORF3 protein after expression and purification is utilized to establish a serological detection method for distinguishing whether the ORF3 of the duck circovirus type 1 is deficient or not.

Disclosure of Invention

The invention aims to provide an ELISA detection kit for detecting whether the ORF3 of the duck circovirus type 1 is deleted or not, application thereof and establishment of an ELISA detection method for detecting whether the ORF3 of the duck circovirus type 1 is deleted or not. The ELISA detection kit can be used for distinguishing whether the ORF3 of the duck circovirus type 1 is deleted or not.

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

an ELISA detection kit for detecting whether the ORF3 of the type 1 duck circovirus has deletion or not comprises an ELISA plate A coated with the Cap recombinant protein of the type 1 duck circovirus and an ELISA plate B coated with the ORF3 recombinant protein of the type 1 duck circovirus.

The preparation method of the type 1 duck circovirus Cap recombinant protein comprises the following steps:

analyzing a type 1 duck circovirus Cap sequence, optimizing by a codon to obtain an optimized type 1 duck circovirus Cap protein sequence, then synthesizing a corresponding nucleotide sequence in a whole gene, inserting the nucleotide sequence into pET30a, screening to obtain a positive recombinant expression plasmid pET30a-DuCV-1-dCap, 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 type 1 duck circovirus Cap recombinant protein (DuCV-1-dCap protein), wherein the amino acid sequence of the optimized type 1 duck circovirus Cap protein is shown as SEQ ID NO. 1.

The preparation method of the type 1 duck circovirus ORF3 recombinant protein comprises the following steps:

analyzing ORF3 gene of the duck circovirus type 1, carrying out total gene synthesis, inserting the synthesized ORF3 gene into a pET32a expression vector, wherein the expressed ORF3 protein amino acid sequence of the duck circovirus type 1 is shown as SEQ ID NO.2, and screening to obtain a positive recombinant expression plasmid pET32a-DuCV-1-ORF3; transforming the recombinant expression plasmid into a prokaryotic expression competent cell, adopting IPTG to induce and express, and purifying an obtained expression product to obtain the type 1 duck circovirus ORF3 recombinant protein (DuCV-1-ORF 3 protein).

The coating concentration of the type 1 duck circovirus Cap recombinant protein is 2.00 mu g/mL, and the coating concentration of the type 1 duck circovirus ORF3 recombinant protein is 2.50 mu g/mL.

The ELISA detection kit also comprises: coating solution, washing solution, sealant, sample diluent, enzyme-labeled secondary antibody, chromogenic agent, stop solution, negative serum, duCV-1 natural infection positive serum and DuCV-1-delta ORF3 (ORF 3 gene deletion DuCV-1) positive serum.

The coating solution is 0.05M carbonate buffer solution with pH9.6, and the washing solution is 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 detecting whether the ORF3 of the type 1 duck circovirus is deficient or not.

An ELISA detection method for detecting whether the ORF3 of the duck circovirus type 1 is deleted or not comprises the following steps:

(1) Coating: using purified duck circovirus type 1 Cap recombinant protein (DuCV-1-dCap 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 A with 96 holes, coating the antigen on each hole 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 1 ORF3 recombinant protein (DuCV-1-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 to be detected 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 antibody in the incubated hole, washing for 4 times by 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 an enzyme-labeling instrument, and reading the OD value of each sample at 450 nm;

(7) And (4) judging a result:

a) In the indirect ELISA detection method (i.e., duCV-1-dCap-ELISA method) established on the ELISA plate A, when the OD value of the serum to be detected is greater than or equal to 0.499, the serum is judged to be DuCV-1 (no matter whether ORF3 gene is deleted or not) positive, and when the OD value is less than 0.499, the serum is judged to be DuCV-1 negative.

B) An indirect ELISA detection method (namely, a DuCV-1-ORF3-ELISA method) established by the ELISA plate B judges that DuCV-1 (no deletion of ORF3 gene) is positive when the OD value of the serum to be detected is more than or equal to 0.450, and judges that DuCV-1 (no deletion of ORF3 gene) is negative when the OD value is less than 0.450;

c) The same serum was tested positive by DuCV-1-dCap-ELISA and DuCV-1-ORF3-ELISA, and the serum was judged positive for DuCV-1 (natural infection, no deletion of ORF3 gene).

D) The same serum was determined to be positive by DuCV-1-dCap-ELISA, but negative by DuCV-1-ORF3-ELISA, and the serum was determined to be positive by DuCV-1- Δ ORF3 (ORF 3 gene deletion).

The DuCV-1-dCap-ELISA method and the DuCV-1-ORF3-ELISA method are combined to distinguish whether the ORF3 of the duck circovirus type 1 has deletion or not.

In the step (1), the recombinant protein of the envelope antigen type 1 duck circovirus Cap is diluted to 2.00 mu g/mL by using 0.05M carbonate buffer solution with pH9.6; the antigen 1-coated duck circovirus ORF3 recombinant protein is diluted to 2.50 mu g/mL by using a carbonate buffer solution with 0.05MpH9.6. Namely, the envelope concentration of the type 1 duck circovirus Cap recombinant protein is 2.00 mu g/mL, and the envelope concentration of the type 1 duck circovirus ORF3 recombinant protein is 2.50 mu g/mL.

In the step (3), the serum to be detected is treated with PBS buffer according to the ratio of 1: diluting by 80 times;

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 (5) diluting by 4000 times.

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

the invention uses the Cap protein and ORF3 protein of DuCV-1 as envelope antigens to respectively establish a DuCV-1 indirect ELISA detection method, and can be used for researching whether the ORF3 of the duck circovirus type 1 (DuCV-1) has deletion after combination. The method establishes DuCV-1-dCap-ELISA and DuCV-1-ORF3-ELISA serological detection methods. Wherein, duCV-1-dCap-ELISA detects positive serum of DuCV-1 natural infection (i.e. ORF3 is not deleted) and positive serum of DuCV-1 ORF3 gene deletion strain (DuCV-1-delta ORF 3) infection. DuCV-1-ORF3-ELISA was positive for serum positive to DuCV-1 infection (ORF 3 was not deleted), but negative for serum positive to DuCV-1 infection with ORF3 gene-deleted strain (DuCV-1-. DELTA.ORF 3). DuCV-1-dCap-ELISA and DuCV-1-ORF3-ELISA are combined to establish a method for distinguishing whether ORF3 of the duck circovirus type 1 is deleted or not and provide a related ELISA detection kit, and no related research is found at home and abroad at present.

In addition, the detection method of the invention also has the advantages of rapid and convenient detection.

Drawings

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

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

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

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

FIG. 5 is a specific analysis of DuCV-1-dCap-ELISA;

FIG. 6 is a specific analysis of DuCV-1-ORF 3-ELISA.

Detailed Description

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

the first embodiment is as follows: preparation of type 1 duck circovirus Cap recombinant protein (DuCV-1-dCap protein) and type 1 duck circovirus ORF3 recombinant protein (DuCV-1-ORF 3 protein):

prokaryotic expression of 1 type duck circovirus (DuCV-1) Cap protein

1.1 codon optimization

Analysis was performed on the sequence of the Cap protein of DuCV-1, and the optimized amino acid sequence was (the underlined region is the tag sequence on the vector):

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAP KYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHM DSPDLGTDDDDKAMADIGSEFMRGRTYRRAYRGRRKRRSLRRRFRRRRLRIARPRRRFSVVTYKVTRNTVFGFFGSQTGPTAAGKWQSLSLEDGAQYTDPPARGNNICGLNMRWAMFGDTNSYMTGSTPNYHYPYDYYMIKGVAITLRPAYNIYQKSKTQGSTVIDKDGQIVKTATTGWSIDPYGSTSSRRTWDPSRVHRRYFVPKPIIQGAGEGTKYSTFFLGGRNFTWINCTQDQVVHYGMGMSLRKPDNTTGVNAQYDIEAQFTFYIKFGQFTGF

after the corresponding nucleotide sequence is synthesized into related sequence by whole gene, the related sequence is inserted into pET32a, and positive recombination expression plasmid (pET 32 a-DuCV-1-dCap) is obtained after screening.

1.2 protein inducible expression

Transforming the recombinant expression plasmid into a prokaryotic expression competent cell, and adopting IPTG to induce and express, wherein the method specifically comprises the following steps: the recombinant expression plasmid is transformed into prokaryotic expression competent cell Rosetta (DE 3), and a single colony of an 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 0.5mM, and the medium was cultured at 220rpm for 4 hours at 37 ℃. 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 target protein was approximately 47.74kDa in size, 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 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 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 DuCV-1-dCap protein is shown in FIG. 2 (the size of the protein of interest is about 47.74kDa, shown by the arrow in FIG. 2). The concentration of the purified DuCV-1-dCap protein was 0.30mg/ml.

2 DuCV-1-ORF3 protein prokaryotic expression

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-1, according to an 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-DuCV-1-ORF 3) is obtained after screening, wherein an expressed DuCV-1-ORF3 amino acid sequence is shown as follows, and an underlined region is a label on the vector.

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAP KYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHM DSPDLGTDDDDKAMADIGSMSLRPDQRIDPWRLAHQLEGMSTLWRNILHYLHQIPGHERARLFLPATPQGRQLARYFEGSGIPEGEAYHSRPSPRRR

2.2 protein inducible expression

The recombinant expression plasmid is transformed into prokaryotic expression competent cell Rosetta (DE 3), and a single colony of an 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, and the medium was cultured at 220rpm for 4 hours at 37 ℃. 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 target protein was approximately 26.91kDa in size, 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 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 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 Western Blot analysis of purified DuCV-1-ORF3 protein is shown in FIG. 4 (the size of the protein of interest is approximately 26.91kDa, as indicated by the arrow in FIG. 1). The concentration of the purified DuCV-1-ORF3 protein was 1.00mg/ml.

The second embodiment: establishment of indirect ELISA detection method

3 establishment of ELISA method

(1) Coating: using purified duck circovirus type 1 Cap recombinant protein (DuCV-1-dCap protein) as a coating antigen, diluting the recombinant protein with a carbonate buffer solution with the pH value of 0.05M and 9.6, adding the diluted protein into an ELISA plate A with 96 holes, coating the solution on each hole 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 1 ORF3 recombinant protein (DuCV-1-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, and 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: 100 mu.L of serum diluted with PBS buffer is added into each hole of the ELISA plate A and the ELISA plate B, and the mixture is incubated 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 antibody in the incubated hole, washing for 4 times by 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 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:

a) In the indirect ELISA detection method (i.e., duCV-1-dCap-ELISA method) established by the ELISA plate A, the serum OD value to be detected is determined as DuCV-1 (no matter whether ORF3 gene is deleted or not) positive when being more than or equal to 0.499, and the OD value is less than 0.499 and is determined as DuCV-1 negative.

B) An indirect ELISA detection method (namely, a DuCV-1-ORF3-ELISA method) established by the ELISA plate B judges that DuCV-1 (no deletion of ORF3 gene) is positive when the OD value of the serum to be detected is more than or equal to 0.450, and judges that DuCV-1 (no deletion of ORF3 gene) is negative when the OD value is less than 0.450;

c) The same serum was tested positive by DuCV-1-dCap-ELISA and DuCV-1-ORF3-ELISA, and the serum was judged positive for DuCV-1 (natural infection, absence of ORF3 gene deletion).

D) The same serum was determined to be positive by DuCV-1-dCap-ELISA, but negative by DuCV-1-ORF3-ELISA, and the serum was determined to be positive by DuCV-1- Δ ORF3 (ORF 3 gene deletion).

The DuCV-1-dCap-ELISA method and the DuCV-1-ORF3-ELISA method are combined to distinguish whether the ORF3 of the duck circovirus type 1 has deletion or not.

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 envelope antigen type 1 duck circovirus Cap recombinant protein and the type 1 duck circovirus ORF3 recombinant protein are respectively 2.00 mu g/mL and 2.50 mu g/mL, and the concentration of the serum is 1: 80-fold dilution and 1-fold 4000-fold dilution of enzyme-labeled secondary antibody.

The OD values of 30 DuCV-1 negative sera were determined by the established DuCV-1-dCap-ELISA method and the mean value was determined

And Standard Deviation (SD), the mean plus 3 times the standard deviation being taken as the positive decision threshold->

The threshold value is 0.499, that is, if the OD value of the serum to be detected is not less than 0.499, the DuCV-1 is determined to be positive, and if the OD value is less than 0.499, the serum is determined to be negative.

OD values of 30 DuCV-1 negative sera were measured by the established DuCV-1-ORF3-ELISA method and the average value was determined

And Standard Deviation (SD), the mean plus 3 times the standard deviation being considered as a positive decision-making threshold>

The threshold value is 0.450, namely DuCV-1 is judged to be positive if the OD value of the serum to be detected is more than or equal to 0.450, and the OD value is less than 0.450, and the serum is judged to be negative. Note that ORF3 gene-deleted strain DuCV-1 (DuCV-1. DELTA. ORF 3) did not express ORF3, and no positive ORF3 antibody could be detected by DuCV-1-ORF3-ELISA method, while DuCV-1. DELTA. ORF 3-positive serum was negative by DuCV-1-ORF3-ELISA method.

4 specific analysis of ELISA method

4.1 Specificity of DuCV-1-dCap-ELISA

The OD values of the positive sera of the strains DuCV-1 (DuCV-1. DELTA. ORF 3) lacking the AIV, ATmV, GPV, GHPyV, duCV-1 and ORF3 genes were simultaneously determined by the DuCV-1-dCap-ELISA method established above. As shown in FIG. 5, only DuCV-1 and DuCV-1. Delta. ORF 3-infected sera were positive in detection result; positive sera of other pathogens, all negative (see fig. 5).

4.2 Specificity of DuCV-1-ORF3-ELISA

The OD values of AIV, ATmV, GPV, GHPyV, duCV-1 and DuCV-1 gene deletion strain (DuCV-1. DELTA. ORF 3) positive sera were simultaneously measured by the DuCV-1-ORF3-ELISA method established above, and the results are shown in FIG. 5, in which only DuCV-1 test was positive, and positive sera of other pathogens and DuCV-1. DELTA. ORF3 infected sera were negative (see FIG. 6).

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 as the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.

Claims (10)

1. An ELISA detection kit for detecting deletion of ORF3 of type 1 duck circovirus is characterized in that: the enzyme-linked immunosorbent assay kit comprises an enzyme-linked immunosorbent assay plate A coated with type 1 duck circovirus Cap recombinant protein and an enzyme-linked immunosorbent assay plate B coated with type 1 duck circovirus ORF3 recombinant protein.

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

analyzing a Cap protein sequence of the duck circovirus type 1, optimizing by a codon to obtain an optimized duck circovirus type 1 Cap protein amino acid sequence, then wholly synthesizing a corresponding nucleotide sequence, inserting the nucleotide sequence into pET30a, screening to obtain a positive recombinant expression plasmid pET30a-DuCV-1-dCap, transforming the recombinant expression plasmid into a prokaryotic expression competent cell, carrying out IPTG (isopropyl-beta-thiogalactoside) induced expression, and purifying an obtained expression product to obtain the duck circovirus type 1 Cap recombinant protein, wherein the optimized duck circovirus type 1 Cap protein amino acid sequence is shown as SEQ ID NO. 1.

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

after ORF3 gene of the duck circovirus type 1 is analyzed, the complete gene is synthesized into related nucleotide sequence and inserted into pET32a expression vector, the expressed ORF3 protein amino acid sequence of the duck circovirus type 1 is shown as SEQ ID NO.2, and positive recombinant expression plasmid pET32a-DuCV-1-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 type 1 duck circovirus ORF3 recombinant protein.

4. The ELISA detection kit of claim 1, wherein: the coating concentration of the type 1 duck circovirus Cap recombinant protein is 2.00 mu g/mL, and the coating concentration of the type 1 duck circovirus ORF3 recombinant protein is 2.50 mu g/mL.

5. The ELISA detection kit of claim 1, wherein: it still includes:

the ELISA detection kit also comprises: coating solution, washing solution, sealant, sample diluent, enzyme-labeled secondary antibody, chromogenic agent, stop solution, negative serum, duCV-1 natural infection positive serum and DuCV-1-delta ORF3 (ORF 3 gene deletion DuCV-1) positive serum.

6. The use of the ELISA detection kit of any one of claims 1-5 for detecting the presence or absence of a deletion in ORF3 of duck circovirus type 1.

7. An ELISA detection method for detecting whether ORF3 of type 1 duck circovirus is deleted or not is characterized in that: the method comprises the following steps:

(1) Coating: taking purified type 1 duck circovirus Cap recombinant protein as a coating antigen, diluting with carbonate buffer solution with 0.05MpH9.6, adding into an ELISA plate A with 96 holes, coating at 4 ℃ overnight at 100 mu L per hole; 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 type 1 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 to an ELISA plate B with 96 holes, coating each hole with 100 mu L of the antigen 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;

(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 to be detected 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 antibody in the incubated hole, washing for 4 times by 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 an enzyme-labeling instrument, and reading the OD value of each sample at 450 nm;

(7) And (4) judging a result:

a) In the indirect ELISA detection method (i.e., duCV-1-dCap-ELISA method) established on the ELISA plate A, when the OD value of the serum to be detected is greater than or equal to 0.499, the serum is judged to be DuCV-1 (no matter whether ORF3 gene is deleted or not) positive, and when the OD value is less than 0.499, the serum is judged to be DuCV-1 negative.

B) An indirect ELISA detection method (namely, a DuCV-1-ORF3-ELISA method) established by the ELISA plate B judges that DuCV-1 (no deletion of ORF3 gene) is positive when the OD value of the serum to be detected is more than or equal to 0.450, and judges that DuCV-1 (no deletion of ORF3 gene) is negative when the OD value is less than 0.450;

c) The same serum was tested positive by DuCV-1-dCap-ELISA and DuCV-1-ORF3-ELISA, and the serum was judged positive for DuCV-1 (natural infection, no deletion of ORF3 gene).

D) The same serum was determined to be positive by DuCV-1-dCap-ELISA, but negative by DuCV-1-ORF3-ELISA, and this serum was determined to be positive by DuCV-1- Δ ORF3 (ORF 3 gene deletion).

The DuCV-1-dCap-ELISA method and the DuCV-1-ORF3-ELISA method are combined to distinguish whether the ORF3 of the duck circovirus type 1 has deletion or not.

8. The ELISA detection method for detecting the absence or absence of deletion of ORF3 of type 1 duck circovirus according to claim 7, wherein the ELISA detection method comprises the following steps: in the step (1), the envelope antigen 1 type duck circovirus Cap recombinant protein is diluted to 2.00 mu g/mL by using a carbonate buffer solution with the pH value of 0.05MpH9.6; the antigen 1-coated duck circovirus ORF3 recombinant protein is diluted to 2.50 mu g/mL by using 0.05M carbonate buffer solution with pH9.6.

9. The ELISA detection method for the presence or absence of deletion of ORF3 of type 1 duck circovirus according to claim 7, wherein the ELISA detection method comprises the following steps: in the step (1), the preparation method of the type 1 duck circovirus Cap recombinant protein comprises the following steps:

analyzing a Cap protein sequence of the duck circovirus type 1, optimizing by a codon to obtain an optimized Cap protein amino acid sequence of the duck circovirus type 1, synthesizing a corresponding nucleotide sequence in a whole gene, inserting the nucleotide sequence into pET30a, screening to obtain a positive recombinant expression plasmid pET30a-DuCV-1-dCap, transforming the recombinant expression plasmid into a prokaryotic expression competent cell, performing IPTG (isopropyl-beta-thiogalactoside) induced expression, and purifying an obtained expression product to obtain the duck circovirus type 1 Cap recombinant protein, wherein the optimized Cap protein amino acid sequence of the duck circovirus type 1 is shown as SEQ ID NO. 1;

the preparation method of the type 1 duck circovirus ORF3 recombinant protein comprises the following steps:

after ORF3 gene of the duck circovirus type 1 is analyzed, the complete gene is synthesized into related nucleotide sequence and inserted into pET32a expression vector, the expressed ORF3 protein amino acid sequence of the duck circovirus type 1 is shown as SEQ ID NO.2, and positive recombinant expression plasmid pET32a-DuCV-1-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 type 1 duck circovirus ORF3 recombinant protein.

10. The ELISA detection method for detecting the absence or absence of deletion of ORF3 of type 1 duck circovirus according to claim 7, wherein the ELISA detection method comprises the following steps: in the step (3), the serum to be detected is treated with PBS buffer according to the 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.

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