CN114487397A

CN114487397A - Indirect ELISA detection kit for detecting porcine delta coronavirus

Info

Publication number: CN114487397A
Application number: CN202210078648.5A
Authority: CN
Inventors: 黄耀伟; 李亚丽; 王斌; 史芳舒
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-01-24
Filing date: 2022-01-24
Publication date: 2022-05-13

Abstract

The invention discloses an indirect ELISA detection kit for detecting porcine delta coronavirus. The ELISA detection kit established by taking the NS6 protein of the PDCoV as the coating antigen can accurately detect the swine coronavirus antibody in a clinical sample. Meanwhile, the NS6 protein is used as an accessory protein specific to the swine delta-coronavirus, does not have cross reaction with other coronaviruses, and can be used for detecting the antibody of the PDCoV after the swine infects the human or other animals. The ELISA kit established by the protein has the characteristics of strong specificity, high sensitivity and good repeatability, provides a new choice for the rapid diagnosis and immune monitoring of PDCoV, and has important significance for the prevention, control and purification of the disease.

Description

Indirect ELISA detection kit for detecting porcine delta coronavirus

Technical Field

The invention relates to the technical field of biology, in particular to an indirect ELISA detection kit for detecting porcine delta coronavirus.

Background

The delta coronavirus (Deltacoronavirus) belongs to the family Coronaviridae (Coronaviridae) and the subfamily Coronaviridae (Coronavirinae), and is a type of coronavirus which has been newly found in recent years. 2012, (Patrick c.y.wo, et al, Y. (2012). discover of seven novel mammalia and avo-corvoviruses in the gene deltacoavrus supplorts of genes as the gene source of alpharonavirus and avoronavirus and avian coronaviruses as the gene source of gamma-coloronavir and deltacoronavirus.j Virol, 86 (7)) Porcine delta coronavirus (PDCoV) was first found in feces collected from live poultry market. PDCoV can infect the swinery of all ages, causes diarrhea, vomiting, dehydration and other symptoms similar to porcine epidemic diarrhea and porcine transmissible gastroenteritis, the morbidity and the mortality can reach 50% -100%, especially the most serious morbidity of suckling piglets seriously threatens the health of the swinery, and brings huge economic loss to the pig industry.

The PDCoV genome structure is similar to other coronaviruses, the total length is about 25.4kb, and the genome structure encodes replicase ORF1ab, S glycoprotein (S), small molecule membrane protein (E), membrane protein (M) and nucleocapsid protein (N) from 5 '-3' end in sequence. In addition to the common structural proteins common to coronaviruses, there are a number of specific ORFs encoding replication proteins, some of which are incorporated at low levels into the virion. The PDCoV accessory protein gene NS6 (GenBank number of protein: QPI70915.1) has no obvious homology with accessory protein genes of other coronaviruses, is positioned between M and N genes and encodes a protein of 94 amino acids (aa). The predicted molecular weight is 13 kDa. Research shows that virus accessory protein NS6 exists in PDCoV infected cell, and research also proves that accessory protein NS6 is expressed in animal body during infection and is involved in assembly of virus particle.

Experimental infection studies have shown that PDCoV can infect calves, chickens, turkeys and mice. PDCoV infected chickens and turkeys exhibit diarrhea, viral RNA is detectable in cloacal and tracheal discharge, and the distended gastrointestinal tract contains a yellow mixture of liquid and gas (Boley, Patricia A et al, "hormone Deltaconovir Infection and Transmission in Poultry, United states," emulsifying Infection diseases volumes.26, 2 (2020): 255-. In PDCoV-infected Calves, viral RNA shedding is prolonged, but no clinical symptoms or overt enteropathy are exhibited, indicating that PDCoV is of limited infectivity in Calves (Jung, Kwoil et al, "Calves area stable to infection with the new engineered porcine delayed virons, but not t with the bacterial enteric virons. porcine epidemic diarrhe virus." Archives of virology vol.162, 8 (2017): 2357-. Asymptomatic infection of PDCoV in the mouse gut and lymphoid tissues suggests that it may be a host for PDCoV, and be involved in further interspecies transmission (Liu, Yan et al, "rolls of Two Major Domains of the Port Deltaconovir S1 Subunit in Receptor Binding and Neutralization," Journal of virology vol.95, 24 (2021): e 0111821.). In addition, it was found in a recent study (Lednicky, J.A., et al., (2021) Independent infections of porcine deltacoronavir amplitude Haitian childredn. Nature, 600(7887), 133-137.) that porcine delta coronavirus was found in plasma samples of three children with acute febrile disease, which only identified symptomatic children as acute PDCoV infections, and further serological studies were required to determine the frequency of such infections in the general population, but serological studies were somewhat difficult due to the possible cross-reactivity of the virus with human endemic coronavirus.

Disclosure of Invention

In order to overcome the defects in the prior art, the NS6 protein is used as a helper protein special for the swine delta-coronavirus, is not present in other human coronaviruses, does not have cross reaction with other human coronaviruses, and can be used for detecting antibodies of PDCoV from swine infection to human or other animals.

The invention firstly provides application of a swine delta coronavirus NS6 protein as a detection target in preparation of a kit for detecting the swine delta coronavirus. Preferably, the kit is an indirect ELISA detection kit and comprises the swine coronavirus NS6 protein serving as a coating antigen. Preferably, the sample to be tested is a serum sample of human or porcine origin.

The invention also provides an indirect ELISA detection kit for detecting the porcine delta coronavirus, which comprises the porcine delta coronavirus NS6 protein serving as a coating antigen. Preferably, the sample to be tested is a serum sample of human, porcine, bovine, chicken, turkey or mouse origin.

Preferably, the indirect ELISA detection kit further comprises a washing solution, a coating solution, a blocking solution, a sample diluent, a substrate developing solution, a stop solution, a negative control, a positive control and a secondary antibody,

when the sample to be detected is a serum sample from human, pig, cow, chicken, turkey or mouse, the secondary antibody is corresponding to goat/mouse/rabbit anti-human, pig, cow, chicken, turkey or mouse IgG. For example, when the sample to be detected is a serum sample of human origin, the secondary antibody is goat/mouse/rabbit anti-human IgG; when the sample to be detected is a serum sample of porcine origin, the secondary antibody is a goat/mouse/rabbit anti-pig IgG.

The invention also provides an indirect ELISA detection method for detecting the porcine delta coronavirus for non-disease diagnosis, which uses the indirect ELISA detection kit and comprises the following steps:

(1) adding the NS6 protein of the envelope antigen of the swine T-type coronavirus into an ELISA plate, and coating the ELISA plate;

(2) after coating the antigen, adding a sealing liquid into the ELISA plate for sealing;

(3) diluting a sample to be detected, a negative control and a positive control by using a sample diluent, adding the diluted sample, the negative control and the positive control into an enzyme label plate, adding a secondary antibody after incubation, and continuing incubation;

(4) adding substrate developing solution for developing, adding stop solution to stop reaction after reaction in dark place, and detecting OD with enzyme-labeling instrument₄₅₀A value;

(5) judging the result, and judging the result to be positive when the OD450 value of the sample to be detected is more than or equal to 0.377; when OD is reached₄₅₀When the value is less than 0.334, the result is negative; when OD is more than or equal to 0.334₄₅₀The value is suspected to be positive when the value is less than 0.377, the measurement is required to be repeated, and the OD is measured again when the OD is measured again₄₅₀If the value is still more than or equal to 0.334, the result is judged to be positive, otherwise, the result is negative.

The indirect ELISA detection method for detecting the swine T-type coronavirus can be used for detecting whether a sample contains an antibody aiming at the antigen swine T-type coronavirus NS6 protein, so that whether an individual from a sample is infected by PDCoV can be known, and the indirect ELISA detection method can also be used for immune monitoring of the transmission infection condition of the PDCoV.

Preferably, the antigen coating concentration is 50 ng/well/100 uL. Sealing at 37 deg.C for 2 hr; the dilution ratio of serum is 1: 100; the serum incubation time was 30 min. The concentration of the secondary antibody is 1: 10000; the optimal secondary antibody reaction time is 30 min; the optimal substrate development time was 15 min.

The swine coronavirus NS6 protein serving as the envelope antigen is constructed on the basis of a prokaryotic expression vector, and the prokaryotic expression vector for expressing the swine coronavirus NS6 protein is used for constructing the swine coronavirus NS6 protein, so that the process is simple, the expression level is high, and the purification is easy. Moreover, an ELISA detection kit established by taking the swine delta coronavirus NS6 protein prepared by the expression vector provided by the invention as a coating antigen can accurately detect the anti-swine delta coronavirus antibody in a clinical sample. Meanwhile, the kit has the advantages of strong specificity, good repeatability, simplicity, rapidness and low cost, provides a new choice for PDCoV diagnosis and immune monitoring, and has important significance for prevention, control and purification of the disease.

The ELISA detection kit established by taking the NS6 protein of the PDCoV as the coating antigen can accurately detect the porcine delta-coronavirus antibody in a clinical sample. Meanwhile, the NS6 protein is used as an accessory protein specific to the swine delta-coronavirus, does not have cross reaction with other coronaviruses, and can be used for detecting the antibody of the PDCoV after the swine infects the human or other animals. The ELISA kit established by the protein has the characteristics of strong specificity, high sensitivity and good repeatability, provides a new choice for the rapid diagnosis and immune monitoring of PDCoV, and has important significance for the prevention, control and purification of the disease.

Drawings

FIG. 1 is a diagram showing the results of the double digestion of the pCold I-PDCoV-NS6 recombinant plasmid provided in example 1, wherein a lane M: standard molecular weight Marker, lane 1: EcoR I and Xba I were double digested, lane 2: no cleavage, lane 3: EcoRI single enzyme digestion.

FIG. 2 is a graph showing the results of solubility analysis of the expression protein PDCoV-NS6 using SDS-PAGE as provided in example 1, wherein a lane M: standard molecular weight Marker, lane 1: post-induction pCold I-unloaded supernatant, lane 2: pCold I after induction precipitated without load, lane 3: post-induction pCold I-NS6 supernatant, lane 4: pCold I-NS6 precipitated after induction.

FIG. 3 is an identification chart of SDS-PAGE results of the purified PDCoV-NS6 protein provided in example 1, wherein, lane M: standard molecular weight Marker, lane 1: pCold I empty, lane 2: before purification of pCold I-PDCoV-NS6, lane 3: after purification of pCold I-PDCoV-NS 6.

FIG. 4 is a Western blot assay result chart of the PDCoV-NS6 protein provided in example 1, wherein A: the primary antibody is anti-His rabbitt (dilution multiple 1: 5000), and the secondary antibody is coat-anti-rabbitt (dilution multiple 1: 5000); b: the primary antibody is anti-NS6 rabbitt (dilution multiple 1: 100), and the secondary antibody is coat-anti-rabbitt (dilution multiple 1: 5000); c: the primary antibody was anti-NS6 mouse (dilution factor 1: 1000), the secondary antibody was coat-anti-mouse (dilution factor 1: 5000), and A, B, C in each lane M: standard molecular weight Marker, lane 1: pCold I empty, lane 2: pCold I-PDCoV-NS 6.

FIG. 5 is a graph showing the results of determination of cut off by indirect ELISA as provided in example 9.

FIG. 6 is a graph showing the results of the indirect ELISA specificity test provided in example 10.

FIG. 7 is a graph showing the results of the indirect ELISA assay for anti-PDCoV IgG antibodies in clinical pig serum provided in example 12.

Detailed Description

The invention provides an expression vector for expressing a swine T-type coronavirus NS6 protein, which is constructed on the basis of a prokaryotic expression vector and integrates a PDCoV-NS6 gene to obtain the prokaryotic expression vector for expressing the swine T-type coronavirus NS6 protein.

Wherein, the PDCoV-NS6 gene sequence is shown in SEQ ID NO. 1; the amino acid sequence of the PDCoV-NS6 protein is shown as SEQ ID NO. 2.

Compared with other escherichia coli expression vectors, the escherichia coli cold shock expression vector for expressing the swine coronavirus NS6 protein provided by the invention has higher expression efficiency and less foreign protein, and is low in price compared with a baculovirus expression system.

The invention also provides a preparation method of the expression vector, which comprises the following steps:

the PDCoV-NS6 gene is subjected to PCR amplification by taking a PDCoV-NS6 gene (SEQ ID NO.1) as a template and PDCoV-NS6-F (5'-CCGGAATTCATGTGCAACTGCCAT-3') and PDCoV-NS6-R (5'-TGCTCTAGATTAATTTAATTCATCTTC-3') as primers to obtain a PDCoV-NS6 gene with two ends respectively containing EcoR I and Xba I enzyme cutting sites, and the PDCoV-NS6 gene containing the EcoR I and Xba I enzyme cutting sites is cloned to the EcoR I/Xba I site of a prokaryotic expression vector pCold I to obtain the pCold I-PDCoV-NS6 prokaryotic expression vector for expressing the NS6 protein of the swine coronavirus.

In the invention, the method for preparing the PDCoV-NS6 protein by using the expression vector comprises the following steps: and (3) transforming the recombinant expression plasmid into an expression host escherichia coli BL21, collecting bacterial precipitation, and performing affinity purification to obtain the PDCoV-NS6 protein.

The invention also provides a swine T-type coronavirus NS6 protein prepared by applying the prokaryotic expression vector.

The expression purification is carried out based on the prokaryotic expression system provided by the invention, and the expression product of the swine delta coronavirus NS6 protein has the advantages of high expression level, time and labor saving and high purity.

In addition, the invention also provides an indirect ELISA detection kit for the protein of the swine T-type coronavirus NS6, wherein the envelope antigen is the protein of the swine T-type coronavirus NS6 which is expressed and purified based on the prokaryotic expression system provided by the invention.

In the invention, the kit further comprises a washing solution, a coating solution, a blocking solution, a sample diluent, a substrate developing solution, a stop solution, a negative control, a positive control and a secondary antibody.

Wherein the coating liquid is a product of Solarbio company; the washing solution is PBS + 0.05% Tween-20; the diluent is PBS + 0.05% Tween-20+ 5% skimmed milk powder; the confining liquid is PBS + 0.05% Tween-20+ 5% skimmed milk powder; the substrate color developing solution is a single component TMB substrate developing solution (Solarbio company); the stop solution is 2M H₂SO₄A solution; the negative control is PDCoV negative pig serum, and the positive control is PDCoV positive serum; the secondary antibody is goat anti-pig IgG marked by horseradish peroxidase.

The operation program of the indirect ELISA kit provided by the invention comprises the following steps:

step (1): adding the coating antigen into the ELISA plate at the amount of 50 ng/hole/100 mu L, and coating the ELISA plate;

step (2): after the coating is carried out overnight, adding a sealing liquid into the ELISA plate for sealing;

and (3): adding the diluted sample, the negative control and the positive control into an enzyme label plate, adding a second antibody after incubation, and continuing incubation;

adding substrate color developing solution for color development, adding stop solution to stop reaction after reaction in a dark place, and measuring OD of each hole by using an enzyme-labeling instrument₄₅₀A value;

judging the result in the step (5), and judging the result to be positive when the OD value is more than or equal to the average value X +3 SD; when the OD value is suspected to be positive between X +2SD and X +3SD, retesting is required; and judging the test result to be positive when the retest value is still larger than X + SD, otherwise, judging the test result to be negative.

The indirect ELISA detection kit established by taking the NS6 protein as the coating antigen can accurately detect the IgG antibody of the anti-porcine delta-coronavirus, and meanwhile, the antigen quantity of the NS6 protein used by the kit is as low as 50 ng/hole/100 mu L, namely, about 50 mu g of antigen is needed by a 96-hole plate, so that the cost is greatly saved compared with 200 ng/hole by taking the N protein as the coating antigen. In conclusion, the kit has the advantages of strong specificity, high sensitivity, simple process and low cost, and can be used for clinical application.

Example 1 construction of prokaryotic expression System and preparation of PDCoV-NS6 protein

Construction of expression vector for NS6 Gene

1.1 primer design

NS6 fragment is amplified by using DNAstar software design primer according to NS6 gene sequence of swine T-coronavirus strain (Genbank Accession Number: MW196362.1), wherein an upstream primer PDCoV-NS6-F sequence is 5'-CCGGAATTCATGTGCAACTGCCAT-3', and a downstream primer PDCoV-NS6-R sequence is 5'-TGCTCTAGATTAATTTAATTCATCTTC-3'. The primers were synthesized by Shanghai Biometrics, Inc.

1.2 construction of pCold I-PDCoV-NS6 recombinant plasmid

The artificially synthesized PDCoV-NS6 gene fragment and the pCold I vector are respectively subjected to double enzyme digestion by EcoR I and Xba I, and are connected to construct pCold I-PDCoV-NS6 expression plasmid. The correctly identified recombinant plasmids (as shown in FIG. 1) were sent to Shanghai Biopsis for sequence determination and alignment by software.

The recombinant plasmid pCold I-PDCoV-NS6 with correct sequencing is subjected to double enzyme digestion identification, enzyme digestion is carried out for 2h at the temperature of EcoR I and Xba I37 ℃, meanwhile, the non-enzyme digestion plasmid and the single enzyme digestion plasmid are used as a control, and the result is shown in figure 1, which indicates that the initial construction of the recombinant plasmid is successful.

1.3 prokaryotic expression of PDCoV-NS6 protein

1) Transforming the recombinant plasmid with correct sequencing into an expression host escherichia coli BL21, plating, selecting a single colony, inoculating into an LB culture medium containing 50mmol/L, and culturing at 37 ℃ and 200rpm overnight;

2) expanding culture to mid-log phase, i.e. measuring OD₆₀₀When the value is about 0.6, adding IPTG with the final concentration of 1mmol/L to induce and culture overnight at the temperature of 16 ℃ and the rpm of 180;

3) taking a proper amount of expressed thalli, carrying out ultrasonic crushing in ice bath, respectively taking thalli cracking supernatant and sediment to carry out SDS-PAGE electrophoresis, and analyzing the solubility of expressed protein;

4) placing the induced expression bacterial liquid in 12000 Xg, centrifuging for 15min, discarding supernatant, collecting thalli precipitate, and carrying out heavy suspension by using 1 XPBS;

5) repeating the steps again, carrying out ultrasonic crushing in ice bath, and pausing for 3s when the power P is 40% ultrasonic for 3 s;

6) collecting the crushed sample, placing the sample at 4 ℃, centrifuging the sample at 12000 Xg for 30min, respectively taking a proper amount of supernatant and sediment, adding a loading buffer into the supernatant and the sediment, and preparing a sample for SDS-PAGE detection.

1.4 identification of PDCoV-NS6 protein solubility: taking a proper amount of bacteria liquid after induction expression, centrifuging, then discarding supernatant, and collecting precipitate; PBS is resuspended, centrifugated after ultrasonication, the supernatant and the precipitate are collected, and loading buffer is respectively added for SDS-PAGE analysis of protein solubility. Meanwhile, the empty vector bacteria solution was used as a control. The identification result is shown in FIG. 2, and the result shows that the PDCoV-NS6 protein exists in the form of inclusion bodies. Purification of PDCoV-NS6 protein

1) Single clones were amplified into 350mL LB medium, followed by sonication and lysis with 70mL Buffer A overnight at 4 ℃.

2) Centrifuging the thallus lysate at 12000 Xg at 4 deg.C for 30min, collecting supernatant, and removing thallus precipitate; undissolved impurities were removed with a 0.45 μm filter.

3) Pretreating Ni-NTA, adding appropriate amount of filler into protein purification column, standing for 20min, sequentially adding ddH₂O, Binding buffer, then transferred to centrifuge tubes containing the samples and combined overnight at 250rpm on a shaker at 4 ℃.

4) The treated mixture was loaded into a protein purification column, left to stand for 10min, and then allowed to flow down slowly (one drop for 3s to 5 s), collecting the liquid, named flow-through.

5) Eluting with 30mL of Buffer B, Buffer C and Buffer D in sequence, eluting with imidazole of different concentrations to remove heteroprotein, and collecting imidazole eluates of different concentrations for Coomassie brilliant blue staining.

6) According to the result of SDS-PAGE gel, selecting the eluent with the highest protein concentration for concentration, and selecting a 3kDa ultrafiltration concentration tube for concentration because the size of the PDCoV-NS6 protein is predicted to be 13 kDa; it is rinsed with double distilled water before use, and centrifuged at 3000 Xg for 30 min.

7) Replacing protein with 6M, 4M and 2M urea, centrifuging at 3000 Xg for 15min, collecting the replaced liquid, replacing 4M urea when the volume reaches 1/3 of the original volume, and repeating;

8) carrying out protein concentration on the eluent by using a protein displacement tube and displacing imidazole in the eluent;

9) the concentration of the protein after concentration and replacement is determined by using a BCA kit, and the operation steps refer to the instruction.

SDS-PAGE analysis and WB analysis of the purified PDCoV-NS6 protein:

SDS-PAGE analysis of the purified protein was performed, and the control was performed with the vector before purification and the vector empty, respectively, and the results are shown in FIG. 3, showing that the amount of the foreign protein was reduced. And Western blot analysis is carried out by using a His label antibody and anti-PDCoV rabbit and mouse polyclonal antiserum respectively, the result is shown in figure 4, the size of an expression product is consistent with the predicted size of NS6 protein, and in each small graph, a lane 1 is an empty carrier bacterial liquid cracking sediment, and a lane 2 is a pCold I-PDCoV-NS6 bacterial liquid cracking sediment.

The PDCoV recombinant NS6 protein obtained by the embodiment has good expression, high purity and good reactivity, and can be used for subsequent experiments.

Example 2 procedure for Indirect ELISA method

(1) Antigen coating

The protein PDCoV-NS6 provided by the embodiment 1 of the invention is used as antigen and diluted to 50 ng/hole/100 mu L by using a coating buffer solution, then the protein PDCoV-NS6 is added into an enzyme label plate, is taken out overnight (12 h-14 h) at 4 ℃, liquid in the hole is discarded, PBST is washed for 3 times, each time is 3min, and the plate is dried;

(2) sealing of

Blocking solution (150. mu.L/well) was added to the plate and incubated at 37 ℃ for 120 min. Taking out, discarding the blocking solution, washing with PBST for 3 times, 3min each time, and drying;

(3) sample application

The samples were subjected to 1: 100 dilution, adding to the enzyme label plate, 100u L/hole, 37 degrees C were incubated for 30 min. Taking out, removing liquid in the hole, washing with PBST for 3 times, 3min each time, and drying;

(4) adding a second antibody

The secondary antibody is diluted by 1: 10000 by using a confining liquid, added into an enzyme label plate, and incubated for 30min at the temperature of 37 ℃ at a rate of 100 mu L/hole. Taking out, removing liquid in the hole, washing with PBST for 3 times, 3min each time, and drying;

(5) adding substrate for color development

Adding single-component TMB color development liquid into an enzyme label plate, carrying out a light-shielding reaction at 37 ℃ for 15min at a concentration of 100 mu L/hole;

(6) termination of the reaction

Adding 2M H into enzyme label plate₂SO₄Stop solution, 50 μ L/well, measuring with enzyme-labeling instrument within 10min after adding, and recording OD of each well₄₅₀A value;

(7) interpretation of results

When the OD value is more than or equal to the average value X +3SD, judging the result as positive; when the OD value is suspected to be positive between X +2SD and X +3SD, retesting is required; and judging the test result to be positive when the retest value is still larger than X +2SD, otherwise, judging the test result to be negative.

Example 3 optimization of optimal coating concentration of recombinant NS6 protein and optimal working concentration of Secondary antibody

The recombinant NS6 protein was sequentially diluted with coating solution to 6 dilutions of 4. mu.g/mL, 2. mu.g/mL, 1. mu.g/mL, 0.5. mu.g/mL, 0.25. mu.g/mL, and 0.125. mu.g/mL, each dilution coating 1 vertical row of the ELISA plate. HRP-labeled goat anti-pig IgG antibody is sequentially diluted by 1: 2000, 1: 4000, 1: 5000, 1: 8000, 1: 10000 and 1: 20000 times by using antibody diluent, and each dilution is added in the same horizontal line at a rate of 100 mu L/hole to form a square matrix. The positive serum OD value is about 1.0, the negative serum OD450 value is less than 0.3, and the antigen concentration and the secondary antibody dilution with the maximum ratio (P/N value) of the positive serum OD value to the negative serum OD value are selected as the optimal antigen coating concentration and the optimal secondary antibody reaction concentration.

TABLE 1 determination of antigen coating concentration and dilution factor of secondary antibody

The result of the square matrix titration shows that: when the antigen coating concentration is 0.5 mug/mL and the concentration of the enzyme-labeled secondary antibody is 1: 10000, the P/N value obtained by detection is as high as 5.67, which is the best detection effect. Thus, the optimal working concentration of antigen was 0.5. mu.g/mL, and the optimal dilution factor of the secondary antibody was 1: 10000 (Table 1).

EXAMPLE 4 determination of optimum blocking conditions

The optimal recombinant protein antigen coating concentration, 100 mu L/hole and the optimal enzyme-labeled secondary antibody dilution degree. The ELISA plates were divided into four groups. The first group was blocked at 37 ℃ for 1h, the second group at 37 ℃ for 2h, the third group at 37 ℃ for 3h, and the fourth group at 37 ℃ for 4 h. After washing, ELISA reaction was performed. Each group was assayed for positive and negative serum OD₄₅₀And calculating the P/N value, wherein the optimal blocking time is determined when the positive serum OD value is close to 1.0, the negative serum OD value is less than 0.3 and the P/N value is maximum.

TABLE 2 determination of optimal blocking conditions

The results are shown in Table 2, and considering all together, blocking at 37 ℃ for 2h is the optimum blocking condition.

EXAMPLE 5 determination of optimal serum dilution

The optimal recombinant protein antigen coating concentration is adopted, 100 mu L/hole is washed and then sealed for 2h at 37 ℃, and then the negative and positive serum diluted by 1: 100, 1: 200, 1: 400, 1: 800, 1: 1600 and 1: 3200 are respectively added, 100 mu L/hole is added, and each dilution is repeated for 3 times. Respectively carrying out ELISA reaction, and respectively measuring the average OD of negative and positive serum in each serum reaction time after the reaction is finished₄₅₀And calculating the P/N value to determine the optimal serum dilution factor.

TABLE 3 determination of serum dilution

As a result, as shown in Table 3, the P/N value was the largest when the serum dilution was 1: 100, and therefore the optimal serum dilution was 1: 100.

Example 6 determination of optimal serum incubation time

Coating with optimal recombinant protein antigen concentration at 100 μ L/well, washing, sealing at 37 deg.C for 2 hr, adding 1: 100 diluentThe released negative and positive serum is divided into four groups with 100 μ L/well, the first group reacts at 37 deg.C for 30min, the second group at 37 deg.C for 60min, the third group at 37 deg.C for 90min, and the fourth group at 37 deg.C for 120min, each group is repeated for 3 times. Respectively carrying out ELISA reaction, and respectively measuring the average OD of negative and positive serum in each serum reaction time after the reaction is finished₄₅₀And calculating the P/N value to determine the optimal serum incubation time.

TABLE 4 determination of optimal serum incubation time

As shown in Table 4, the P/N value was the largest when the serum incubation time was 30min (0.5h), and the optimal serum incubation time was 30 min.

Example 7 determination of optimal Secondary antibody reaction time

The optimal recombinant protein antigen coating concentration is 100 mu L/hole. Washing, sealing for 2h, adding 1: 100 times diluted negative and positive serum, 100 μ L/well, reacting at 37 deg.C for 30min, washing, adding 1: 10000 times diluted HRP-labeled goat anti-pig IgG antibody, 100 μ L/well. The reaction time is divided into four groups, wherein the first group reacts at 37 ℃ for 15min, the second group reacts at 37 ℃ for 30min, the third group reacts at 37 ℃ for 45min, the fourth group reacts at 37 ℃ for 60min, and each group is repeated for 3 times. Washing, developing, and measuring OD of positive and negative serum on enzyme-linked immunosorbent assay₄₅₀And calculating the P/N value to determine the optimal reaction time of the secondary antibody.

TABLE 5 determination of optimal Secondary antibody reaction time

As a result, as shown in Table 5, it was found that the maximum P/N value was obtained when the OD value of the positive serum was close to 1.0 and the OD value of the negative serum was less than 0.3, when the secondary antibody reaction time was 30 min. Therefore, the optimal secondary antibody reaction time was determined to be 30 min.

EXAMPLE 8 determination of the development time of the substrate

The optimal recombinant protein antigen coating concentration is 100 mu L/hole, after washing, the solution is sealed for 2h at 37 ℃, and then the negative and positive serum diluted by 1: 100 times is added, 100 mu L/hole, and the reaction is carried out for 30min at 37 ℃. After washing, HRP-labeled goat anti-pig IgG antibody diluted 1: 10000 times is added, 100 mu L/hole, and reaction is carried out at 37 ℃ for 30 min. After washing, the product was divided into four groups. Adding 100 μ L/hole substrate color development solution, and developing at 37 deg.C for 5 min; adding 100 μ L/hole substrate color development solution into the second group, and developing at 37 deg.C for 10 min; adding 100 μ L/hole substrate color development solution, and developing at 37 deg.C for 15 min; and adding 100. mu.L/well substrate color development solution into the fourth group, and developing at 37 ℃ for 20 min. Repeating each group for 3 times, adding 50 μ L/well stop solution to stop color development after color development is completed, and respectively detecting OD of each group of positive and negative serum₄₅₀And calculating the P/N value to determine the optimal substrate color development time.

TABLE 6 determination of substrate reaction time

As a result, as shown in Table 6, it was found that the P/N value was the largest at development time of 15min, and the optimum substrate development time was 15 min.

Example 9 determination of indirect ELISA cut off value

48 pig negative sera were taken and tested by an established and optimized indirect ELISA method. The antigen coating concentration is 0.5 mug/mL; the concentration of the enzyme-labeled secondary antibody is 1: 10000; sealing at 37 deg.C for 2 hr; the dilution ratio of serum is 1: 100; the incubation time of the serum is 30 min; the optimal secondary antibody reaction time is 30 min; the optimal substrate development time was 15 min.

By statistical analysis, negative serum OD₄₅₀The mean (X) of the values was 0.248, and the variance was 0.043, so the Cut off value was mean +3SD of 0.377. Namely, when the OD450 is more than or equal to 0.377, the judgment is positive; when OD is measured₄₅₀When the value is less than 0.334(mean +2SD), the result is judged to be negative; while the suspect interval is determined, i.e., when 0.334≤OD₄₅₀If the value is less than 0.377, the result is suspected to be positive, and the test is required to be repeated; and when the retest value is still more than or equal to 0.334, judging the result to be positive, otherwise, judging the result to be negative. The determination of the Cut off value is shown in FIG. 5.

EXAMPLE 10 specificity test

The enzyme label plate is coated by purified recombinant protein PDCoV NS6 protein, standard positive serum of porcine epidemic diarrhea virus, porcine enterocoronavirus positive serum and porcine transmissible gastroenteritis virus positive serum are detected under the same condition according to an established and optimized ELISA method, PDCoV negative and positive serum contrast is set at the same time, and whether cross reaction with antibodies of other pathogens exists is judged by taking the established PDCoV NS6 protein indirect ELISA antibody detection method as a standard. The results show that the OD of the porcine epidemic diarrhea virus-positive serum and the porcine enteric coronavirus-positive serum₄₅₀The values are all smaller than cut off value (see FIG. 6), thus demonstrating that the indirect ELISA established by the research on the PDCoV-NS6 protein antibody has good specificity.

EXAMPLE 11 repeatability test

In-batch repeatability test: at the same time, the same enzyme label plate is coated with PDCoVNS6 protein, 3 times are set, and OD is determined₄₅₀And calculating the average value and the standard deviation, and according to the formula: coefficient of variation (CV%) [ standard deviation (S)/mean (X) ]]X 100%, calculating the variation coefficient in the batch, and determining the repeatability in the batch.

TABLE 7 repeatability test in batches

The results are shown in Table 7, which indicate that the variation coefficient of the repeatability within the batch ranges from 0.27% to 3.73%, and the repeatability within the batch is higher.

Batch to batch repeatability test: coating PDCoV NS6 protein on enzyme label plates at different time, performing ELISA detection on positive and negative serum, and determining OD₄₅₀And calculating the average value and the standard deviation, and according to the formula: coefficient of variation (CV%) [ standard deviation (S)/mean (X) ]]X 100%, calculating inter-batch variation coefficient, and determiningBatch-to-batch repeatability.

TABLE 8 repeatability tests between batches

The results are shown in Table 8, which indicate that the coefficient of variation for the batch-to-batch repeatability ranged from 0.39% to 8.9%, and that the batch-to-batch repeatability was better.

Example 12 preliminary application of PDCoV NS6 protein Indirect ELISA detection method

Indirect ELISA of PDCoV NS6 protein established in this study was used to detect 679 portions of pig serum samples collected in 2014-2015 from different age stages in shandong, south river, west jiang, south hu, junsu, black dragon and zhejiang provinces, and the level of PDCoVIgG antibodies in the herd at each stage was analyzed, with the results shown in fig. 7. Wherein the seropositive rate of the backup sow reaches 100 percent, the seropositive rate of the first-born sow reaches 98.3 percent, the seropositive rate of the multiparous sow reaches 100 percent, the seropositive rate of the 0-1 week-old piglet reaches 95.6 percent, the seropositive rate of the 3 week-old piglet reaches 78.3 percent, the seropositive rate of the 5 week-old piglet reaches 83.9 percent, the seropositive rate of the 11 week-old weaned piglet is 84.6 percent, the seropositive rate of the 17 week-old weaned piglet is 100 percent, the seropositive rate of the 20 week-old weaned piglet is 97.8 percent, and the seropositive rate of the 679 part of seropositive rate is 92.9 percent.

In conclusion, the experimental results show that the ELISA detection kit consisting of the PDCoV-NS6 protein can accurately, quickly, effectively and specifically detect the anti-porcine delta-coronavirus antibody in a clinical sample.

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Claims

1. Application of the protein NS6 of the porcine delta coronavirus as a detection target in preparing a kit for detecting the porcine delta coronavirus.

2. The use according to claim 1, wherein the kit is an indirect ELISA test kit comprising the porcine coronavirus NS6 protein as a coating antigen.

3. Use according to claim 1, characterized in that the sample to be tested is a serum sample of human, porcine, bovine, chicken, turkey or mouse origin.

4. An indirect ELISA detection kit for detecting porcine delta coronavirus, which is characterized by comprising a porcine delta coronavirus NS6 protein serving as a coating antigen.

5. The indirect ELISA detection kit of claim 4 wherein the sample to be detected is a human or porcine serum sample.

6. The indirect ELISA detection kit of claim 5 further comprising a washing solution, a coating solution, a blocking solution, a sample diluent, a substrate developing solution, a stop solution, a negative control, a positive control and a secondary antibody,

when the sample to be detected is a serum sample from human, pig, cow, chicken, turkey or mouse, the secondary antibody is corresponding to goat/mouse/rabbit anti-human, pig, cow, chicken, turkey or mouse IgG.

7. An indirect ELISA detection method for detecting porcine delta coronavirus for non-disease diagnostic purposes, wherein the indirect ELISA detection kit of claim 6 is used, and wherein the indirect ELISA detection method comprises the following steps:

8. The indirect ELISA detection method of claim 7 wherein the antigen coating concentration is 50 ng/well/100 μ L.

9. The indirect ELISA detection method of claim 7 wherein the blocking conditions are blocking at 37 ℃ for 2 h; the dilution ratio of serum is 1: 100; the serum incubation time was 30 min.

10. The indirect ELISA detection method of claim 7 wherein the concentration of secondary antibody is 1: 10000; the optimal secondary antibody reaction time is 30 min; the optimal substrate development time was 15 min.