Digital coding magnetic bead, kit and method for detecting porcine reproductive and respiratory syndrome virus antibody

Abstract

The invention belongs to the field of biological detection, and relates to a digital coding magnetic bead, a kit and a method for detecting a porcine reproductive and respiratory syndrome virus antibody. The invention provides a detection method capable of qualitatively and quantitatively detecting target molecules simultaneously only by using a small amount of samples, and compared with the traditional technology, the method has the advantages that the detection precision and sensitivity are improved, the cost is reduced, the specificity and expansibility are high, the test result is visual, and the detection accuracy is high by matching with special fluorescence detection and photographing equipment. The method can play an important role in large-scale centralized detection and government supervision, and has a very wide market prospect.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and relates to digitally encoded magnetic beads, a kit and a method for detecting porcine reproductive and respiratory syndrome virus antibodies. Background Art

[0002] At present, the microchip coding technology mainly includes fluorescent coding and digitally coded magnetic beads (Barcoded Magnetic Beads, BMB) technology. Fluorescent coding is the most commonly used technology at present, and its representative is the fluorescent biological microspheres produced by Luminex Corporation in the United States. Its principle is that the fluorescent biological microspheres mixed with three fluorescent dyes in different proportions are used as reaction carriers, and the fluorescence of different wavelengths excited by dyes in different proportions is identified as the basis for identifying different microspheres. This technology is widely used, but affected by the reaction principle and reaction environment, the consistency of the test results is poor and the test cost is high, so its current commercial application is relatively limited. Digital digital coding technology is a new biochip technology platform based on microchip coding technology. Because it is not affected by fluorescent substances, it is a completely digital biological digital coding technology. It has more accurate recognition capabilities and can detect 4096 digital microchip indicators at a time. It is a revolutionary progress in microchip coding technology.

[0003] The present invention adopts digital digitally encoded magnetic bead technology as a detection technology platform. This technology uses a white LED light source to identify the digital code of the digitally encoded magnetic bead chip, and uses a green LED light source to detect the reporter fluorescence on the reporter molecule on a single magnetic bead chip. The white LED light source can classify the magnetic bead chips, thereby identifying different reaction types (i.e. qualitative); the green LED light source can determine the number of reporter fluorescent molecules bound to the magnetic bead chip, thereby determining the number of target molecules bound to the magnetic bead chip (i.e. quantitative), and superimpose the two sets of CCD rapid imaging, and perform rapid qualitative and quantitative analysis through software. Therefore, through the simultaneous detection of white and green LED dual light sources, real-time, qualitative and quantitative analysis of the reaction is completed. ( Figure 1 , Figure 2 ) The original experimental image includes the orientation of the magnetic bead chip, the two-dimensional code, the number of magnetic bead chips and the fluorescence intensity information ( Figure 3 ).

[0004] The binding reaction of antigen-antibody, enzyme substrate, ligand receptor and nucleic acid hybridization reaction are carried out on digital magnetic beads with different codes. The microchip code and reporter fluorescence are detected by two different laser beams to achieve qualitative and quantitative purposes. Up to hundreds of different biological reactions can be completed in one reaction well. During the detection, the sample and reporter molecule (for example, phycoerythrin) are added successively to react with the labeled microchip. The target molecule (antigen or antibody to be detected, etc.) in the sample can specifically bind to the labeled microchip to form a microchip-target molecule-reporter molecule complex. The microchip is then detected and the results are imaged and analyzed ( Figure 4 ).

[0005] Porcine reproductive and respiratory syndrome, also known as blue ear disease, is an important infectious disease caused by the porcine reproductive and respiratory syndrome virus. It can easily cause reproductive disorders in pregnant sows and respiratory symptoms in pigs of all ages. It is highly contagious through contact and has local epidemic characteristics. Pigs of all ages and breeds can be infected with this virus. It is one of the major epidemic diseases currently plaguing the pig industry in my country and the world.

[0006] Commonly used immunological detection methods include immunofluorescence staining, enzyme-linked immunosorbent assay, and colloidal gold method, etc. At present, there are no technical research reports or patents in China for conducting porcine reproductive and respiratory syndrome virus antibody detection through digital coding platforms. Summary of the invention

[0007] In one aspect, the present invention provides an antigenic peptide of porcine reproductive and respiratory syndrome virus, the polypeptide sequence of the antigenic peptide is shown in SEQ ID NO:1.

[0008] In one aspect, the present invention provides the use of the antigenic peptide, which is any one of the following a1-a4: a1. Detection of antibodies against the N protein of porcine reproductive and respiratory syndrome virus; a2. Preparation of products for detecting antibodies to the N protein of porcine reproductive and respiratory syndrome virus; a3. Preparation of products for detecting antibodies to the N protein in animals immunized with porcine reproductive and respiratory syndrome virus; a4. Preparation of products for detecting antibodies against the N protein in animals infected with porcine reproductive and respiratory syndrome virus.

[0009] In one aspect, the present invention provides a kit for detecting antibodies to the N protein of porcine reproductive and respiratory syndrome virus, the kit comprising the antigenic peptide.

[0010] On the one hand, the present invention provides a digitally encoded magnetic bead for detecting antibodies to the N protein of porcine reproductive and respiratory syndrome virus, wherein the digitally encoded magnetic bead is coupled to the antigen peptide.

[0011] On the one hand, the present invention provides a digitally encoded magnetic bead kit for detecting antibodies to the N protein of porcine reproductive and respiratory syndrome virus, the kit comprising: a digitally encoded magnetic bead solution coupled with the antigen peptide; and at least one of a positive control, a negative control, a secondary antibody solution, a sample diluent, a sample processing solution, a color developing solution, an assay buffer, and a reaction microplate.

[0012] In some embodiments, the concentration of the antigen peptide in the digitally encoded magnetic bead solution is 6 µg / 10,000 digitally encoded magnetic beads.

[0013] In some embodiments, the secondary antibody solution is a Sulfo-NHS-LC-LC-Biotin or Sulfo-NHS-LC-Biotin biotin-labeled anti-swine secondary antibody protein solution, with a concentration of 1.5-3µg / mL; preferably, the sample processing solution is a phosphate buffer containing 0.05% Tween-20 and 0.05% ProClin950, with a pH of 7.4; preferably, the sample diluent is a sample processing solution containing 1% BSA; preferably, the color developing solution is a sample diluent containing 2.5~5µg / mL SA-PE; preferably, the positive control is a standard porcine reproductive and respiratory syndrome positive serum; preferably, the negative control is a standard porcine reproductive and respiratory syndrome negative serum. In some embodiments, the reaction microplate is a transparent flat-bottomed plastic 96-well plate.

[0014] On the one hand, the present invention provides a method for detecting antibodies to the N protein of porcine reproductive and respiratory syndrome virus, using the antigen peptide, the kit, the digitally encoded magnetic beads, or the digitally encoded magnetic bead kit for detection.

[0015] In some embodiments, the method comprises the following steps: S1. Collect pig serum samples; S2. Add the porcine serum sample to the sample diluent, dilute it at a ratio of 1:50, and shake it to prepare the sample solution; S3. Use a pipette to draw 50 µL of the digitally encoded magnetic bead solution of porcine reproductive and respiratory syndrome into the reaction microwell, and then continue to add 50 µL of the sample solution treated in step S2; incubate at room temperature for 90 minutes; perform positive and negative controls at the same time; the negative control is tested using standard porcine reproductive and respiratory syndrome negative serum; S4. Wash twice with 150 µL sample treatment solution on a plate washer or manually, add 50 µL secondary antibody solution, and incubate at room temperature for 30 min. S5. Wash twice with 150 µL sample treatment solution on a plate washer or manually, add 50 µL SA-PE color development solution, and incubate at room temperature for 30 min. S6. Wash 4 times on a plate washer or manually with 150 µL of sample treatment solution, and add 200 µL of assay buffer. S7. Use a digital digital coded magnetic bead analyzer to read the digital coded magnetic bead fluorescence signal, and determine the sample detection result according to the fluorescence value of the sample detection, that is, the MFI value.

[0016] In some embodiments, in step S7, when the MFI value of the sample is less than 1235, the test is determined to be invalid and needs to be repeated; when 1235≤MFI value≤1810, the sample is determined to be negative; when the sample MFI value is ≥1810, the sample result is positive.

[0017] In one aspect, the present invention provides a detection system for detecting antibodies to the N protein of porcine reproductive and respiratory syndrome virus in a sample to be tested, the detection system comprising the following components: 1) Detection components for antibodies against the N protein of porcine reproductive and respiratory syndrome virus; 2) Data processing components; 3) Result output component; The detection component of the porcine reproductive and respiratory syndrome virus N protein antibody contains the antigen peptide, the test kit, the digitally encoded magnetic beads, or the digitally encoded magnetic bead test kit.

[0018] In some embodiments, the data processing component is configured to: The sample detection result is determined according to the fluorescence value of the sample to be tested detected by the detection component, that is, the MFI value.

[0019] In some embodiments, when the MFI value of the sample is <1235, the test is determined to be invalid and needs to be repeated; when 1235≤MFI value≤1810, the sample is determined to be negative; when the sample MFI value is ≥1810, the sample result is positive.

[0020] The beneficial effects of the present invention are: The digitally encoded magnetic bead (BMB) detection method for detecting porcine reproductive and respiratory syndrome antibodies provided by the present invention is easy to operate and can be performed without special training. It has high detection sensitivity and is suitable for large-scale and rapid clinical detection. It can also achieve quantitative detection, providing a new technical platform for laboratory diagnosis of porcine reproductive and respiratory syndrome.

[0021] Compared with traditional laboratory testing methods, the present invention has the following advantages: 1) High detection sensitivity and low cost. This method takes advantage of the digitally encoded magnetic bead detection technology and uses fluorescence intensity detection. It has extremely high detection sensitivity and low single detection cost.

[0022] 2) The test results can be subsequently qualitatively and quantitatively determined. A calibration curve can be made using the gradient dilution of the positive control, which can further determine the titer of porcine reproductive and respiratory syndrome virus antibodies in the test sample to achieve quantitative and semi-quantitative determination.

[0023] Compared with traditional technologies, the digitally encoded magnetic bead (BMB) detection method for detecting porcine reproductive and respiratory syndrome antibodies provided by the present invention has outstanding advantages, especially for the strong demand for porcine reproductive and respiratory syndrome antibody detection, and has a very broad market prospect. It provides a new technical platform for large-scale laboratory detection of porcine reproductive and respiratory syndrome virus antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Digital identification of digitally coded magnetic beads (BMB) microchips.

[0025] Figure 2 Fluorescence intensity identification of digitally encoded magnetic beads (BMB) microchips.

[0026] Figure 3 This is an illustration of the image recognition of a digitally coded magnetic bead (BMB) microchip. The first arrow indicates the orientation of the magnetic bead chip (upward or downward); "28" indicates the number of No. 8 magnetic bead chips in the field of view; the number "8" indicates the number of the magnetic bead chip; and "189.6" indicates the fluorescence value of the magnetic bead chip.

[0027] Figure 4 Schematic diagram of the principle of protein (antigen / antibody) detection using digitally encoded magnetic beads (BMB) microchip.

[0028] Figure 5 This is a schematic diagram of the recognition effect of polypeptide sequence a under the condition of positive serum dilution 1:50 in Example 1.

[0029] Figure 6 This is a schematic diagram of the recognition effect of polypeptide sequence b under the condition of positive serum dilution 1:50 in Example 1.

[0030] Figure 7 This is a schematic diagram of the recognition effect of polypeptide sequence c under the condition of positive serum dilution 1:50 in Example 1.

[0031] Figure 8 This is a schematic diagram of the recognition effect of polypeptide sequence d under the condition of positive serum dilution 1:50 in Example 1. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0033] Example 1 Screening and preparation of antigenic protein The viral N protein is the only component of the PRRSV nucleocapsid protein. During the virus infection of cells, the N protein is expressed at the highest level and is produced earliest. It has the advantages of strong immunogenicity and strong conservation. Its gene is about 372bp long.

[0034] 1. Peptide sequence design. Download representative PRRSV virus strain sequences from GenBank, align the N protein conserved regions using DNAStar, and design the following four groups of peptide sequences: a.VRHHFTPSERQLCLS (SEQ ID NO: 1); b.QKRKKGDGQPVNQLC (SEQ ID NO: 2); c.QGAGTCTLSDSGRISYTVEFSL (SEQ ID NO: 3); d.QNQSRGKGPGKKN (SEQ ID NO: 4); 2. Prokaryotic expression and purification. Taking into account the expression and purification aspects, pET-28a(+) (Novagen) was selected as the expression vector. Prokaryotic expression was adopted. The target gene was connected to the expression vector by double enzyme digestion and ligase to form a recombinant plasmid, which was transformed into competent cells. After screening and identification on the selective culture medium, expression was induced and finally purified.

[0035] 3. Antigen polypeptide sequence screening. After prokaryotic expression and purification, the four groups of polypeptide sequences were diluted 1:50 with positive serum, and the implementation scheme of this patent was carried out on the four groups of polypeptide sequences to verify its effect. Each test was repeated three times, and the fluorescence values ​​of the results are shown in the following table.

[0036] Table 1 Fluorescence values ​​of different peptide sequences reacting with serum

[0037] From the table above, we can see that the peptide sequence a is positive when the positive serum is diluted 1:50 (see Figure 5 ); When the positive serum was diluted 1:50, the fluorescence values ​​of peptide sequence b and peptide sequence d were significantly lower than those of peptide sequence a (see Figure 6 , 8 ); The test result of polypeptide sequence c was negative when the positive serum was diluted 1:50 times (see Figure 7 ). It can be seen that the peptide sequence a has the highest fluorescence value and performs best, so the peptide sequence a is selected as the antigen for the following test.

[0038] Example 2 Preparation of Porcine Reproductive and Respiratory Syndrome Digitally Encoded Magnetic Beads (BMB) 1. Antigen coupling. The digitally encoded magnetic bead microchip is a P-carboxyl microchip purchased from Applied Biocode in the United States. The product number is: 44-B0312. This type of microchip is specially optimized for protein / antibody coupling to improve coupling efficiency and sensitivity. Protein coupling is carried out using the currently common EDC-catalyzed carboxyl amidation reaction. Each reaction reagent is a standard commercial reagent or a manually prepared reagent. The components are shown in Table 2.

[0039] Table 2 Reagent configuration table

[0040] The specific steps are as follows: 1) Add ≤150,000 BMBs to a 2 mL microcentrifuge tube and remove the supernatant. Add 76 µL activation buffer, 14 µL freshly prepared 50 mg / mL Sulfo-NHS, and 10 µL freshly prepared 50 mg / mL EDC and incubate on a shaker at room temperature for 60 min.

[0041] 2) Remove the supernatant and wash twice with 100µL coupling buffer, then add 100µL of antigen (peptide sequence a) diluted with coupling buffer. The antigen dilution is controlled at 4-8µg / 10000BMB and incubate at room temperature on a shaker for 120 minutes.

[0042] 3) Remove the supernatant, wash once with NaCl-Tween buffer, add PBST, and incubate at room temperature on a shaker for 10 min.

[0043] 4) Remove the supernatant, add 50 mM TRIS-HCL pH 7.5 buffer, incubate for 30 min and wash twice.

[0044] 5) Remove the supernatant, incubate with blocking buffer and wash twice.

[0045] 6) Remove the supernatant and wash twice with 500 µL of assay buffer.

[0046] 7) Resuspend the BMB in assay buffer and store the BMB at 2-8°C or perform immunoassay immediately.

[0047] Example 3 Preparation of biotin-labeled anti-swine secondary antibody protein solution 1. Secondary Antibody Preparation The secondary antibody was a general anti-swine secondary antibody commercial reagent purchased from Beijing Baixinyi Biotechnology Co., Ltd. (Batch No. A03B36).

[0048] 2. Desalination Unless otherwise specified, the secondary antibody needs to be desalted. Please refer to the instructions for the corresponding reagents for the desalting steps.

[0049] 3. Biotinylation The secondary antibody biotinylation reaction was performed using Sulfo-NHS-LC-LC-Biotin or Sulfo-NHS-LC-Biotin commercial reagent. The biotinylation reagent was purchased from Thermo (lot number 21338). The specific steps are as follows: 1) Dissolve 1-10 mg of protein (i.e., anti-swine secondary antibody protein) in 0.5-2.0 mL of PBS to obtain a protein solution; 2) Dissolve 2.0 mg of biotinylation reagent in 300 µL of ultrapure water to obtain a 10 mM biotin reagent solution; 3) Add an appropriate amount of 10 mM biotin reagent solution to the protein solution to obtain a reactant; 4) Incubate the reactants on ice for two hours or at room temperature for 30 minutes. At this time, the protein labeling is complete and a biotin-labeled anti-swine secondary antibody protein solution (hereinafter referred to as biotin-labeled secondary antibody or secondary antibody) is obtained.

[0050] Example 4 Preparation of SA-PE color developing solution SA-PE was purchased from Yantai Kress Biotechnology Co., Ltd. (Batch No. 68053). SA-PE was diluted to 2-10 µg / mL with activation buffer and stored at 2-8°C in the dark.

[0051] Example 5 Establishment of digitally encoded magnetic bead antigen coating concentration and secondary antibody dilution The antigen was diluted in 5 gradients of 2 µg / 10000BMB, 4 µg / 10000BMB, 6 µg / 10000BMB, 8 µg / 10000BMB, and 10 µg / 10000BMB, respectively, and coupled to the coated microchip according to Example 2. Each gradient was repeated in two wells, 50 µL / well, and standard positive and negative sera were added and incubated at room temperature for 90 minutes.

[0052] The biotin-labeled secondary antibody was diluted to 1:250, 1:500, 1:1000 and 1:2000, and each dilution was repeated in two wells, 50 µL / well, and incubated at room temperature for 30 minutes. SA-PE colorimetric solution was added and incubated at room temperature in the dark for 30 minutes before termination.

[0053] Read the MFI value of each well and form a square array to determine the optimal coating concentration of the antigen and the dilution of the secondary antibody. Find the antigen concentration and the dilution of the secondary antibody in the reaction well with the highest ratio of the positive serum MFI value to the negative serum MFI value (i.e. P / N) as the optimal antigen coating concentration and dilution of the secondary antibody. (As shown in Table 3 below) Table 3 Square array test

[0054] The optimal coating concentration of antigen was determined to be 6µg / 10000BMB by square array test, and the optimal dilution of secondary antibody was 1:1000.

[0055] Example 6 Determination of serum dilution The test was conducted based on the optimal antigen coating concentration of 6µg / 10000BMB and the optimal secondary antibody dilution of 1:1000. The sample positive serum and negative serum were diluted to 1:25, 1:50, 1:200 and 1:400 in succession. The other conditions remained unchanged. The test results are shown in Table 4.

[0056] Table 4 Establishment of the optimal serum dilution

[0057] The optimal serum dilution was determined to be 1:50 through the serum dilution test.

[0058] Example 7 Determination of Serum Reaction Time Based on the optimal antigen coating concentration of 6µg / 10000BMB, the optimal secondary antibody dilution of 1:1000, and the optimal serum dilution of 1:50, standard positive and negative serum was added for reaction, and divided into 4 groups according to the reaction time, the first group: 22℃ for 30 min; the second group: 22℃ for 60 min; the third group: 22℃ for 90 min; the fourth group 22℃ for 120 min. After the secondary antibody was reacted at 22℃ for 60 min, SA-PE colorimetric solution was added for 30 min, the MFI value of each well was measured, and the P / N value of each group of positive and negative serum was calculated to determine the optimal reaction time of the serum to be tested. The test results are shown in Table 5.

[0059] Table 5 Establishment of the optimal serum reaction time

[0060] Through the serum reaction time test, it was determined that 90 minutes at 22°C is the optimal serum reaction time. If the temperature is lowered, the serum reaction time should be appropriately extended.

[0061] Example 8 Establishment of the reaction time of biotinylated secondary antibody Based on the optimal antigen coating concentration of 6µg / 10000BMB, the optimal secondary antibody dilution of 1:1000, the optimal serum dilution of 1:50, and the optimal serum reaction time of 22℃ for 90min, standard serum was added for reaction, and then biotinylated secondary antibody was added. According to the action time of the secondary antibody, the samples were divided into 4 groups: 22℃ for 30 min; the second group: 22℃ for 45 min; the third group: 22℃ for 60 min; the fourth group: 22℃ for 90 min. After washing, SA-PE colorimetric solution was added, the MFI value of each well was measured, and the P / N value of each group of positive and negative serum was calculated to determine the optimal time for the enzyme-labeled secondary antibody reaction. The test results are shown in Table 6.

[0062] Table 6 Establishment of the optimal secondary antibody reaction time

[0063] The optimal secondary antibody reaction time was determined by the secondary antibody reaction time test at 22°C for 30 min.

[0064] Example 9 CUT OFF value establishment According to the established method, 20 negative serum samples were tested, with 2 replicates per serum sample, and the test results are shown in Table 7.

[0065] Table 7 Determination of negative critical value

[0066] The average MFI of 20 serum samples was calculated to be 1522, with a standard deviation of 96.

[0067] According to statistical principles, it can be seen from Table 7 that when the MFI value of the sample is less than 1235 (negative MFI average value-3SD), the test is invalid and it is recommended to redo it; when 1235≤MFI value≤1810 (negative MFI+3SD), the sample result is judged to be negative; when the sample MFI value is greater than 1810, the sample result is judged to be positive.

[0068] Example 10 Sensitivity test Four standard positive sera and four standard negative sera for porcine reproductive and respiratory syndrome virus were selected respectively, and gradient dilutions were performed at 1:200, 1:400, 1:800, and 1:1600. The commercial porcine reproductive and respiratory syndrome antibody detection kit (Batch No.: CPA.99w7 of Hibolai Biofactory, Spain) and the porcine reproductive and respiratory syndrome digitally encoded magnetic bead analysis detection method established by the present invention were used for simultaneous detection.

[0069] The test results show that the porcine reproductive and respiratory syndrome digitally encoded magnetic bead analysis and detection method established by the present invention can still effectively detect when the sample is diluted to 1:1600; the commercial kit cannot effectively detect when the sample is diluted to 1:1600. It can be seen that the method established by the present invention is more sensitive than the traditional ELISA method, and the test results are shown in Table 8.

[0070] Table 8 Sensitivity test

[0071] Example 11 Specificity Test According to the established operating procedures, positive sera of four major diseases causing pig reproductive disorders, namely, classical swine fever (CSF), pseudorabies (PPR), porcine parvovirus (PPV), and foot and mouth disease (FMD), were used for detection. The results are shown in Table 9. The positive sera for detecting these four diseases had no cross-reaction with this test, indicating that the present invention has good specificity.

[0072] Table 9 Specificity test

[0073] Example 12 Repeatability Test Four different batches of coated digitally encoded magnetic beads were prepared, and one known positive serum was tested. Six replicates were set for each batch, and the inter-batch and intra-batch coefficients of variation CV = (SD / MFI average) × 100% were calculated. The test results are shown in Table 10. The intra-batch and inter-batch coefficients of variation were both less than 10%, indicating that the present invention has good repeatability.

[0074] Table 10 Repeatability test

Claims

1. An antigenic peptide of porcine reproductive and respiratory syndrome virus, characterized in that: The polypeptide sequence of the antigen peptide is shown in SEQ ID NO:

1.

2. The use of the antigen peptide according to claim 1, characterized in that: Any one of the following a1-a4: a1. Detection of antibodies against the N protein of porcine reproductive and respiratory syndrome virus; a2. Preparation of products for detecting antibodies to the N protein of porcine reproductive and respiratory syndrome virus; a3. Preparation of products for detecting antibodies to the N protein in animals immunized with porcine reproductive and respiratory syndrome virus; a4. Preparation of products for detecting antibodies against the N protein in animals infected with porcine reproductive and respiratory syndrome virus.

3. A kit for detecting antibodies to the N protein of porcine reproductive and respiratory syndrome virus, characterized in that: The kit comprises the antigen peptide according to claim 1.

4. A digitally encoded magnetic bead for detecting antibodies to the N protein of porcine reproductive and respiratory syndrome virus, characterized in that: The digitally encoded magnetic beads are coupled with the antigen peptide as claimed in claim 1.

5. A digitally encoded magnetic bead kit for detecting antibodies to the N protein of porcine reproductive and respiratory syndrome virus, characterized in that: The kit comprises: a digitally encoded magnetic bead solution coupled with the antigen peptide as claimed in claim 1.

6. The digitally encoded magnetic bead kit according to claim 5, characterized in that: The kit comprises at least one of a positive control, a negative control, a secondary antibody solution, a sample diluent, a sample processing solution, a color developing solution, a determination buffer solution, and a reaction microplate.

7. The digitally encoded magnetic bead kit according to claim 5, characterized in that: In the digitally encoded magnetic bead solution, the antigen peptide concentration is 4-10 µg / 10000 digitally encoded magnetic beads.

8. A method for detecting antibodies to the N protein of porcine reproductive and respiratory syndrome virus, characterized in that: The detection is performed using the antigen peptide according to claim 1, the kit according to claim 3, the digitally encoded magnetic beads according to claim 4, or the digitally encoded magnetic bead kit according to any one of claims 5-7.

9. A detection system for detecting antibodies to the N protein of porcine reproductive and respiratory syndrome virus in a sample to be tested, characterized in that: The detection system comprises the following components: 1) Detection components for antibodies against the N protein of porcine reproductive and respiratory syndrome virus; 2) Data processing components; 3) Result output component; The detection component of the porcine reproductive and respiratory syndrome virus N protein antibody contains the antigen peptide as described in claim 1, the kit as described in claim 3, the digitally encoded magnetic beads as described in claim 4, or the digitally encoded magnetic bead kit as described in any one of claims 5-7.

10. The detection system according to claim 9, characterized in that: The data processing component is configured to: Determine the sample detection result according to the fluorescence value of the sample to be tested detected by the detection component, that is, the MFI value; When the MFI value of the sample is less than 1235, the test is judged to be invalid and needs to be repeated; when 1235≤MFI value≤1810, the sample result is judged to be negative; when the MFI value of the sample is greater than 1810, the sample result is judged to be positive.

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