阅读说明：本技术 猪圆环病毒2型抗原或抗体胶体金快速检测试纸条及方法 (Test paper strip and method for rapidly detecting porcine circovirus type 2 antigen or antibody colloidal gold ) 是由 卓洵辉 丁豪杰 丁建祖 陆绍红 孔庆明 陈睿 郑斌 童群波 楼涤 于 2019-10-30 设计创作，主要内容包括：本发明公开了一种猪圆环病毒2型抗原或抗体胶体金快速检测试纸条及方法。包括PVC板、样品垫、金标垫、硝酸纤维素膜、吸水垫、C线和T线,胶体金试纸条中采用金标蛋白CAP重组蛋白,为去核定位信号的优势抗原表位肽并带有His标签,通过原核表达系统低温诱导表达至上清液；标记于T线(抗原检测试纸条)或C线(抗体检测试纸条)的抗猪圆环病毒2型CAP重组蛋白多克隆兔血清,通过CNBr活化琼脂糖凝胶FF偶连CAP重组蛋白后纯化处理。本发明能快速检测猪血清中的抗原或抗体,敏感性高、特异性强,有较好的稳定性,操作简单,能较好地满足不同层次的需求,易于向基层推广。(The invention discloses a test strip and a method for rapidly detecting porcine circovirus type 2 antigen or antibody colloidal gold. The colloidal gold test strip adopts gold-labeled protein CAP recombinant protein which is dominant epitope peptide of a denucleation positioning signal and is provided with a His label, and is expressed to supernatant through low-temperature induction of a prokaryotic expression system; anti-porcine circovirus type 2 CAP recombinant protein polyclonal rabbit serum marked on a T line (antigen detection test strip) or a C line (antibody detection test strip) is coupled with CAP recombinant protein through CNBr activated sepharose FF and then purified. The kit can quickly detect the antigen or the antibody in the pig serum, has high sensitivity, strong specificity, better stability and simple operation, can better meet the requirements of different levels, and is easy to popularize to the basic level.)

1. A colloidal gold test strip for detecting porcine circovirus type 2 antigen or antibody comprises a PVC plate, a sample pad, a gold label pad, a nitrocellulose membrane, a water absorption pad, a C line and a T line, wherein the water absorption pad is arranged at the upper end of the PVC plate and is used as a handheld part; the middle end of the PVC plate is a reaction area, a nitrocellulose membrane is arranged on the reaction area, a gold label pad, a C line and a T line are arranged on the nitrocellulose membrane, the C line is a quality control line, and the T line is a detection line; the lower end of the PVC plate is provided with a sample pad which is used as an area of a serum sample to be detected; the method is characterized in that: the gold-labeled pad is adsorbed with gold-labeled protein; when the test strip is used for detecting antigen colloidal gold, the gold-labeled protein adsorbed on the gold-labeled pad is porcine circovirus type 2 CAP recombinant protein labeled by colloidal gold, the T line on the nitrocellulose membrane layer is polyclonal rabbit serum resisting the porcine circovirus type 2 CAP recombinant protein, and the C line is goat anti-rabbit IgG polyclonal antibody; when the test strip is used for antibody colloidal gold detection, the gold-labeled protein adsorbed on the gold-labeled pad is porcine circovirus type 2 CAP recombinant protein labeled by colloidal gold, the T line on the nitrocellulose membrane layer is protein A recombinant protein, and the C line is polyclonal rabbit serum resisting the porcine circovirus type 2 CAP recombinant protein.

2. The reagent strip for detecting porcine circovirus type 2 antigen or antibody colloidal gold according to claim 1, which is characterized in that: the porcine circovirus type 2 CAP recombinant protein is obtained by removing a core positioning signal, carrying His label, and carrying out low-temperature induction expression on the recombinant protein of supernatant by using a prokaryotic expression system, and finally purifying through a nickel column, wherein the porcine circovirus type 2 CAP recombinant protein is specifically combined with porcine circovirus type 2 positive serum.

3. The reagent strip for detecting porcine circovirus type 2 antigen or antibody colloidal gold according to claim 1, which is characterized in that: the polyclonal rabbit serum for resisting the porcine circovirus type 2 CAP recombinant protein is obtained by collecting rabbit serum after immunizing a New Zealand white rabbit with the porcine circovirus type 2 CAP recombinant protein, coupling the rabbit serum with the porcine circovirus type 2 CAP recombinant protein through CNBr activated sepharose FF, and purifying the coupled rabbit serum.

4. The reagent strip for detecting porcine circovirus type 2 antigen or antibody colloidal gold according to claim 1, which is characterized in that: the test strip uses the judgment standard as follows:

when the T line and the C line can be clearly seen, the T line and the C line are red, the lines are single, clear and light in background color, and then the detection result of the serum sample to be detected is judged to be positive;

when the T line cannot be clearly seen but the C line can be clearly seen, and the C line is red, the detection result of the serum sample to be detected is judged to be negative;

and if the T line and the C line cannot be clearly seen, namely the T line and the C line are not red, judging that the detection result of the serum sample to be detected is invalid.

5. The reagent strip for detecting porcine circovirus type 2 antigen or antibody colloidal gold according to claim 1, which is characterized in that: the porcine circovirus type 2 CAP recombinant protein is prepared by the following method:

1) design of primers and PCR amplification:

four primer sequences of two pairs of primers are designed and synthesized by utilizing a PCV2 sequence, and PCR amplification is carried out by taking the recombinant plasmid pMD-PCV as a template, wherein the four primer sequences are as follows:

cap-F1: 5-ctggatccaatggcatcttcaacacccgc-3 as shown in SEQ ID NO. 1;

cap-R1: 5-agcagggccagaattcaaccttaacctttc-3 as shown in SEQ ID NO. 2;

cap-F2: 5-ggttgaattctggccctgctccccaatcac-3 as shown in SEQ ID NO. 3;

cap-R2: 5-cccaagctttcacttagggttaagtggggg-3 as shown in SEQ ID NO. 4;

firstly, respectively carrying out first round PCR amplification on two groups of which one group is cap-F1 and cap-R1 and one group is cap-F2 and cap-R2, respectively, carrying out hot start at 94 ℃ for 5 minutes, denaturation at 94 ℃ for 45 seconds, annealing at 60 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, circulating for 30 times, and finally extension at 72 ℃ for 10 minutes;

then mixing two products obtained by two groups of PCR amplification by the mass of 1:1 solution to be used as a template of a second round, carrying out fusion PCR amplification of the second round by taking F1 and R2 as primers, carrying out denaturation at 94 ℃ for 45 seconds, annealing at 60 ℃ for 30 seconds, extending at 72 ℃ for 60 seconds, circulating for 20 times, and finally extending at 72 ℃ for 10 minutes;

finally obtaining a final PCR amplification product;

2) constructing a prokaryotic expression vector and performing induced expression:

recovering the final PCR amplification product by using a gel recovery test strip, carrying out double digestion on the pET32a plasmid and the final PCR amplification product by using BamH I and Hind III, then connecting and transforming the plasmid and the final PCR amplification product to BL21 competent cells, selecting a monoclonal colony, adding the colony into 1mM IPTG after propagation, and carrying out low-temperature induction expression in a shaking table at 16 ℃ to obtain a culture medium;

3) and (3) purifying the recombinant protein:

centrifuging the culture medium for 5 minutes at 8000g of centrifugal force, collecting bacterial liquid, and adding 7ml of PBS for heavy suspension; and (3) carrying out ultrasonic crushing until bacterial liquid is clear, centrifuging for 5 minutes at 8000g of centrifugal force, collecting supernatant, and finally separating and purifying target protein from the supernatant through a nickel column.

6. The reagent strip for detecting porcine circovirus type 2 antigen or antibody colloidal gold according to claim 1, which is characterized in that: the polyclonal rabbit serum for resisting the porcine circovirus type 2 CAP recombinant protein is prepared by the following steps: using porcine circovirus type 2 CAP recombinant protein obtained by separation and purification of a nickel column as an antigen, immunizing 2 New Zealand white rabbits by a multipoint injection method for 1 time every 2 weeks for 3 times, immunizing for the first time with a Freund's complete adjuvant, then immunizing for the second time with a Freund's incomplete adjuvant, collecting blood from the heart after 10 days of last immunization, and separating serum; then, coupling the CNBr activated sepharose FF with the porcine circovirus type 2 CAP recombinant protein, and purifying through the sepharose to obtain immune rabbit serum containing an IgG antibody of the specificity anti-porcine circovirus type 2 CAP recombinant protein.

7. The preparation method of the reagent strip for detecting the porcine circovirus type 2 antigen or antibody colloidal gold, which is applied to any one of claims 1 to 6, is characterized by comprising the following steps:

(1) preparing a colloidal gold solution: 5ml of 1% HAuCl₄Quickly adding into 500ml boiling water, adding sodium citrate 0.1125g, heating for 5min, adjusting rotation speed to cool the solution to 50 deg.C, and sealing at 4 deg.C;

(2) preparing a gold-labeled protein solution: adding 12 mul of K with the concentration of 0.2mol/L into each 1ml of colloidal gold solution₂Adding a CO3 solution and 10 mu g of porcine circovirus type 2 CAP recombinant protein prepared by nickel column separation and purification, adding 10% Bovine Serum Albumin (BSA) solution to make the final concentration of the protein 1%, continuing stirring and oscillating for 30min, then standing for 2h in a refrigerator at 4 ℃, collecting the gold-labeled protein through differential centrifugation, dissolving the gold-labeled protein with a buffer solution at 4 ℃ and storing to obtain a gold-labeled protein solution;

(3) preparing a test strip: respectively arranging a sample pad and a water absorption pad at two ends of a PVC (polyvinyl chloride) plate, arranging a nitrocellulose membrane between the sample pad and the water absorption pad, sequentially arranging a gold-labeled pad, a T line and a C line on the nitrocellulose membrane from the sample pad to the water absorption pad, adsorbing a gold-labeled protein solution diluted by 1:5 on the gold-labeled pad, and sealing the nitrocellulose membrane for 1 h; wherein, the T line of the test strip for detecting the antibody is arranged to be protein A recombinant protein with the concentration of 4mg/ml, and the C line is arranged to be polyclonal rabbit serum of anti-porcine circovirus type 2 CAP recombinant protein with the concentration of 0.5 mg/ml; the test strip T line for detecting the antigen is arranged to be polyclonal rabbit serum of anti-porcine circovirus type 2 CAP recombinant protein with the concentration of 0.5mg/ml, and the C line is arranged to be goat anti-rabbit IgG polyclonal antibody.

8. The method for preparing the reagent strip for detecting porcine circovirus type 2 antigen or antibody colloidal gold according to claim 7, which is characterized in that:

the PVC board below the sample pad is provided with a detection hole, and the detection hole is used for adding a serum sample to be detected.

9. The use method of the colloidal gold test reagent strip for the porcine circovirus type 2 antigen or antibody according to any one of claims 1 to 6, which is characterized by comprising the following steps:

(1) and (3) detection procedures: diluting a serum sample to be detected by using a sample diluent, adding 100 mu l of a stock solution of the serum sample to be detected, diluted according to a volume ratio of 1:50, diluted according to a volume ratio of 1:100 and diluted according to a volume ratio of 1:200 into sample areas at the lower ends of different test strips, standing for 5-10min, and observing;

(2) and (4) judging a result: in each test strip for sample detection, the following judgment method is adopted:

when the T line and the C line can be clearly seen, the T line and the C line are red, the lines are single, clear and light in background color, and then the detection result of the serum sample to be detected is judged to be positive;

when the T line cannot be clearly seen but the C line can be clearly seen, and the C line is red, the detection result of the serum sample to be detected is judged to be negative;

and if the T line and the C line cannot be clearly seen, namely the T line and the C line are not red, judging that the detection result of the serum sample to be detected is invalid.

Technical Field

The invention belongs to a test strip of a diagnosis technology in the technical field of biology, and particularly relates to a colloidal gold test strip for detecting porcine circovirus type 2 (PCV2) antigen or antibody, a preparation method and a use method thereof.

Background

Porcine circovirus type 2 (PCV2) is a main pathogen causing Postwelting Multisystemic Wasting Syndrome (PMWS) of pigs and weaned pigs, and causes symptoms of progressive wasting, slow growth, dyspnea and the like of affected animals. In addition, PCV2 is also related to pig dermatitis and diseases such as nephrotic syndrome, hyperplastic necrotizing pneumonia, pig respiratory disease syndrome, reproductive disorders and the like. Related diseases caused by PCV2 infection are widely distributed in the world, the death rate of pigs is different from 10% to 30%, and serious economic loss is caused to the pig industry.

The main detection methods for porcine circovirus type 2 adopted at home and abroad include virus isolation culture, polymerase chain reaction, indirect immunofluorescence, enzyme-linked immunosorbent assay and the like. These methods can detect the antigen and antibody of PCV2, but have the limitations of long operation time, complex process, need of professional operators and special instruments and equipment, and are difficult to popularize and popularize in the basement. The rapid, simple and accurate detection method is developed, the PCV2 pathogen and antibody level in the swinery are monitored in real time, and the method has important significance for preventing and controlling diseases.

Disclosure of Invention

The invention aims to provide a porcine circovirus type 2 (PCV2) antigen and antibody detection colloidal gold test strip, which can effectively detect an antigen or an antibody in porcine serum and is used for diagnosing the antigen and the antibody infected by PCV 2.

The invention aims to provide a colloidal gold test strip for rapid detection, reduce the detection cost and provide a field diagnosis method capable of being applied in a large scale aiming at the current situations that the PCV2 infection generally causes huge economic loss and lacks an effective field usable test strip in China.

The test paper strip for detecting the porcine circovirus type 2 antigen or antibody colloidal gold is realized by the following technical scheme:

a test paper strip for detecting porcine circovirus type 2 antigen or antibody colloidal gold comprises a PVC plate, a sample pad, a gold label pad, a nitrocellulose membrane, a water absorption pad, a C line and a T line, wherein the water absorption pad is arranged at the upper end of the PVC plate and is used as a handheld part; the middle end of the PVC plate is a reaction area, a nitrocellulose membrane is arranged on the reaction area, a gold label pad, a C line and a T line are arranged on the nitrocellulose membrane, the C line is a quality control line, the T line is a detection line, and the gold label pad, the T line and the C line are sequentially arranged on the nitrocellulose membrane in the direction from the sample pad to the water absorption pad; the lower end of the PVC plate is provided with a sample pad which is used as an area of a serum sample to be detected; the gold-labeled pad is adsorbed with gold-labeled protein; when the test strip is used for detecting antigen colloidal gold, the gold-labeled protein adsorbed on the gold-labeled pad is porcine circovirus type 2 CAP recombinant protein labeled by colloidal gold, the T line on the nitrocellulose membrane layer is polyclonal rabbit serum resisting the porcine circovirus type 2 CAP recombinant protein, and the C line is goat anti-rabbit IgG polyclonal antibody; when the test strip is used for antibody colloidal gold detection, the gold-labeled protein adsorbed on the gold-labeled pad is porcine circovirus type 2 CAP recombinant protein labeled by colloidal gold, the T line on the nitrocellulose membrane layer is protein A recombinant protein, and the C line is polyclonal rabbit serum resisting the porcine circovirus type 2 CAP recombinant protein.

When the test strip is prepared, the porcine circovirus type 2 CAP recombinant protein marked by colloidal gold is adsorbed on a gold-labeled pad; a T line (antigen detection test strip) or a C line (antibody detection test strip) marked on a nitrocellulose membrane by anti-porcine circovirus type 2 CAP recombinant protein polyclonal rabbit serum is assembled by a PVC plate, a sample pad, a gold label pad, the nitrocellulose membrane and a water absorption pad in sequence.

Drawings

FIG. 1 is a graph showing the results of induced expression of recombinant CAP protein.

FIG. 2 is a schematic diagram of a test strip.

Fig. 3 is a diagram illustrating result determination.

FIG. 4 shows the result of the sensitivity test of the antigen test strip.

FIG. 5 is a graph showing the test results of the antibody test strip.

FIG. 6 is a graph showing the test results of the antigen test strip.

After all parts of the test strip are assembled, the test strip is cut into strips with the width of 4mm on a cutting machine, and the strips are sealed and stored for later use, so that the utilization rate of the test strip is improved.

The porcine circovirus type 2 CAP recombinant protein (CAP recombinant protein for short) is a denucleated positioning signal, and is obtained by purifying a genetic engineering recombinant protein which is provided with a His label and is expressed to supernatant by using a prokaryotic expression system through a nickel column at low temperature, wherein the porcine circovirus type 2 CAP recombinant protein is specifically combined with porcine circovirus type 2 positive serum.

The existing detection method of porcine circovirus type 2 antigen or antibody colloidal gold has high requirements on the antigen amount in serum. The invention is provided withPurification by nickel columnThe porcine circovirus type 2 CAP recombinant protein is specially prepared, and an accurate result can still be stably detected under the condition of a smaller amount of antigen in a serum sample to be detected, so that the recognition rate is improved, and the technical problem that the original small amount of antigen cannot be recognized is solved.

The polyclonal rabbit serum for resisting the porcine circovirus type 2 CAP recombinant protein is obtained by collecting rabbit serum after immunizing a New Zealand white rabbit with the porcine circovirus type 2 CAP recombinant protein, coupling the rabbit serum with the porcine circovirus type 2 CAP recombinant protein through CNBr activated sepharose FF, and purifying the coupled rabbit serum.

The test strip uses the judgment standard as follows:

when the T line and the C line can be clearly seen, the T line and the C line are red, the lines are single, clear and light in background color, and then the detection result of the serum sample to be detected is judged to be positive;

when the T line cannot be clearly seen but the C line can be clearly seen, and the C line is red, the detection result of the serum sample to be detected is judged to be negative;

and if the T line and the C line cannot be clearly seen, namely the T line and the C line are not red, judging that the detection result of the serum sample to be detected is invalid.

The porcine circovirus type 2 CAP recombinant protein is prepared by the following method:

1) design of primers and PCR amplification:

four primer sequences of two pairs of primers are designed and synthesized by utilizing a PCV2 sequence, and PCR amplification is carried out by taking the recombinant plasmid pMD-PCV as a template, wherein the four primer sequences are as follows:

cap-F1: 5-ctggatccaatggcatcttcaacacccgc-3 as shown in SEQ ID NO. 1;

cap-R1: 5-agcagggccagaattcaaccttaacctttc-3 as shown in SEQ ID NO. 2;

cap-F2: 5-ggttgaattctggccctgctccccaatcac-3 as shown in SEQ ID NO. 3;

cap-R2: 5-cccaagctttcacttagggttaagtggggg-3 as shown in SEQ ID NO. 4;

the cap-F1 and the cap-R2 are respectively provided with BamH I and Hind III enzyme cutting sites.

The concentration of the solutions of cap-F1, cap-R1, cap-F2 and cap-R2 is 0.5 mu mol/mu l.

Firstly, respectively carrying out first round PCR amplification on two groups of which one group is cap-F1 and cap-R1 and one group is cap-F2 and cap-R2, respectively, carrying out hot start at 94 ℃ for 5 minutes, denaturation at 94 ℃ for 45 seconds, annealing at 60 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, circulating for 30 times, and finally extension at 72 ℃ for 10 minutes;

then mixing two products obtained by two groups of PCR amplification by the mass of 1:1 solution to be used as a template of a second round, carrying out fusion PCR amplification of the second round by taking F1 and R2 as primers, carrying out denaturation at 94 ℃ for 45 seconds, annealing at 60 ℃ for 30 seconds, extending at 72 ℃ for 60 seconds, circulating for 20 times, and finally extending at 72 ℃ for 10 minutes;

finally obtaining a final PCR amplification product;

2) constructing a prokaryotic expression vector and performing induced expression:

carrying out mass recovery on the final PCR amplification product by using a gel recovery test strip, carrying out double digestion on a pET32a plasmid and the final PCR amplification product by using BamH I and Hind III, then connecting and converting the plasmid and the final PCR amplification product to BL21 competent cells, selecting a monoclonal colony, adding the colony into 1mM IPTG after propagation, and carrying out low-temperature induction expression in a shaking table at 16 ℃ to obtain a culture medium;

3) and (3) purifying the recombinant protein:

centrifuging the culture medium for 5 minutes at 8000g of centrifugal force, collecting bacterial liquid, and adding 7ml of PBS for heavy suspension; and (3) ultrasonically crushing until bacterial liquid is clear, centrifuging for 5 minutes at 8000g of centrifugal force, collecting supernatant, separating and purifying target protein by a nickel column, and verifying the size, purity and antigenicity of the protein by SDS-PAGE and western blot after specific implementation.

According to the invention, an immune program is formulated by utilizing the porcine circovirus type 2 CAP recombinant protein expressed by escherichia coli pronucleus, a polyclonal antibody is prepared by immunizing a New Zealand white rabbit, and the polyclonal antibody is specifically purified by coupling the porcine circovirus type 2 CAP recombinant protein with CNBr activated sepharose FF.

The polyclonal rabbit serum for resisting the porcine circovirus type 2 CAP recombinant protein is prepared by the following steps:

using porcine circovirus type 2 CAP recombinant protein obtained by separation and purification of a nickel column as an antigen, immunizing 2 New Zealand white rabbits by a multipoint injection method for 1 time every 2 weeks for 3 times, immunizing for the first time with a Freund's complete adjuvant, then immunizing for the second time with a Freund's incomplete adjuvant, collecting blood from the heart after 10 days of last immunization, and separating serum; then, coupling the CNBr activated sepharose FF with the porcine circovirus type 2 CAP recombinant protein, and purifying through the sepharose to obtain immune rabbit serum containing an IgG antibody of the specificity anti-porcine circovirus type 2 CAP recombinant protein.

Secondly, a preparation method of the porcine circovirus type 2 antigen or antibody colloidal gold test reagent strip, wherein the test strip is prepared as follows:

(1) preparing a colloidal gold solution: 5ml of 1% HAuCl₄Quickly adding into 500ml boiling water, adding sodium citrate 0.1125g, heating for 5min, adjusting rotation speed to cool the solution to 50 deg.C, and sealing at 4 deg.C;

(2) preparing a gold-labeled protein solution: adding 12 mul of K with the concentration of 0.2mol/L into each 1ml of colloidal gold solution₂CO3 solution and 10 mu g of porcine circovirus type 2 CAP recombinant prepared by nickel column separation and purificationAdding 10% Bovine Serum Albumin (BSA) solution to make the final concentration 1%, stirring and oscillating for 30min, standing in a refrigerator at 4 deg.C for 2h, centrifuging at differential speed, collecting gold-labeled protein, dissolving in buffer solution at 4 deg.C, and storing to obtain gold-labeled protein solution;

(3) preparing a test strip: arranging a sample pad and a water absorption pad at two ends of a PVC (polyvinyl chloride) plate respectively, arranging a nitrocellulose membrane between the sample pad and the water absorption pad, wherein two ends of the nitrocellulose membrane are tightly jointed with the sample pad and the water absorption pad respectively, arranging a gold label pad, a T line and a C line on the nitrocellulose membrane in sequence from the sample pad to the water absorption pad, adsorbing a 1:5 diluted gold label protein solution on the gold label pad, and sealing the nitrocellulose membrane for 1 h; wherein, the T line of the test strip for detecting the antibody is arranged to be protein A recombinant protein with the concentration of 4mg/ml, and the C line is arranged to be polyclonal rabbit serum of anti-porcine circovirus type 2 CAP recombinant protein with the concentration of 0.5 mg/ml; the test strip T line for detecting the antigen is arranged into polyclonal rabbit serum of anti-porcine circovirus type 2 CAP recombinant protein with the concentration of 0.5mg/ml, and the C line is arranged into goat anti-rabbit IgG polyclonal antibody;

after all parts of the test strip are assembled, the test strip is cut into strips with the width of 4mm on a cutting machine, and the strips are sealed and stored for later use.

The PVC board below the sample pad is provided with a detection hole, and the detection hole is used for adding a serum sample to be detected.

Thirdly, the application method of the reagent strip for detecting the porcine circovirus type 2 antigen or antibody colloidal gold comprises the following steps:

(1) and (3) detection procedures: diluting a serum sample to be detected by using a sample diluent, adding 100 mu l of a stock solution of the serum sample to be detected, diluted according to a volume ratio of 1:50, diluted according to a volume ratio of 1:100 and diluted according to a volume ratio of 1:200 into sample areas at the lower ends of different test strips, standing for 5-10min, and observing;

(2) and (4) judging a result: in each test strip for sample detection, the following judgment method is adopted:

when the T line and the C line can be clearly seen, the T line and the C line are red, the lines are single, clear and light in background color, and then the detection result of the serum sample to be detected is judged to be positive;

when the T line cannot be clearly seen but the C line can be clearly seen, and the C line is red, the detection result of the serum sample to be detected is judged to be negative;

and if the T line and the C line cannot be clearly seen, namely the T line and the C line are not red, judging that the detection result of the serum sample to be detected is invalid.

The detection sample of the test strip is serum. For whole blood samples, the samples are placed at 37 ℃ for 2 hours or at 4 ℃ overnight, then centrifuged at 1000g for 10 minutes in a centrifuge, and the supernatant is taken for detection.

The colloidal gold test strip can quickly detect the antigen or antibody in the pig serum, the colloidal gold test strip contains the colloidal gold CAP recombinant protein which is the dominant epitope peptide of the denucleation positioning signal and is provided with the His label, and the colloidal gold test strip is subjected to low-temperature induction expression to obtain supernatant through a prokaryotic expression system; polyclonal rabbit serum of anti-porcine circovirus type 2 CAP recombinant protein marked on a T line (antigen detection test strip) or a C line (antibody detection test strip) is obtained by coupling CAP recombinant protein through CNBr activated sepharose FF and purifying.

Compared with the prior art, the invention has the advantages and positive effects that:

firstly, the epitope peptide sequence selected by the invention is a highly conserved segment in PCV2, and has good immunogenicity, so that the detection result has good specificity.

And the anti-CAP rabbit polyclonal antibody used in the invention is a polyclonal antibody which is purified again after the CNBr activated sepharose FF coupling CAP recombinant protein, has stronger capability of identifying and capturing antigens in a sample and better specificity, can identify corresponding antigens in serum with lower antigen content, and has obvious advantages and innovation compared with the test strip which can only detect the antigens in tissues in the prior art.

Thirdly, the antigen detection test strip and the antibody detection test strip are combined for use, so that the infection of the current disease or the past infection can be judged, and the method has practical reference significance for prevention and control of diseases.

Fourthly, the time required by detection is greatly shortened, only 20 minutes are needed at most from the time of obtaining a serum sample to the time of finally obtaining a detection result, other special equipment is not needed, the requirement of detection hardware is lowered, and the method can be widely popularized and used in basic departments.

In summary, the invention has the advantages of high sensitivity, strong specificity, better stability, simple operation, capability of better meeting the requirements of different levels and easy popularization in basic level detection.