A chemiluminescent detection kit for porcine circovirus type 2

Abstract

The invention discloses a porcine circovirus type 2 chemiluminescent detection kit, comprising a polyclonal antibody coupled to magnetic particles, acridinium ester-labeled monoclonal antibody, porcine circovirus type 2 antigen standard solution, chemiluminescent pre-excitation solution A, chemiluminescent excitation solution B, cleaning solution, and a reaction tube. The kit of the invention combines chemiluminescent technology with immunomagnetic particles. Compared with the existing porcine circovirus type 2 detection technology, the kit of the invention has the advantages of high sensitivity, strong specificity, short reaction time, and the like.

Description

Technical Field

[0001] The invention relates to a kit, more specifically, to a porcine circovirus type 2 chemiluminescence detection kit, belonging to the technical field of veterinary disease detection. Background Art

[0002] Porcine circovirus (PCV) is a disease that has attracted much attention in recent years and is widely prevalent around the world. It is a general term for a series of diseases caused by porcine circovirus types. Porcine circovirus belongs to the genus Circovirus and the family Circoviridae. It is one of the smallest known animal viruses. Porcine circovirus type 2 (PCV2) has extremely high pathogenicity. At present, the disease has been widely prevalent in pig herds in my country, causing huge economic losses to the pig industry and seriously endangering the development of the pig industry. It needs to be paid great attention.

[0003] The genome of PCV2 is 1767-1768bp in length, and contains 11 ORFs, encoding proteins of 1.8-35.8kD respectively. ORF1 and ORF2 are the two main open reading frames of PCV2, encoding two proteins. ORF1 encodes virus replication-related protein (Rep), and ORF2 encodes virus capsid protein (Cap). The genome of PCV2 is very small, and the genome is 1767-1768bp in length. PCV2 contains 11 ORFs, encoding proteins of 1.8-35.8kD respectively. ORF1 and ORF2 are the two main open reading frames of PCV2, encoding two proteins in reverse order. ORF1 encodes virus replication-related protein (Rep), and ORF2 encodes virus capsid protein (Cap). ORF2 encodes Cap protein (also known as nucleocapsid protein), which is the main structural protein of the virus and contains the main antigenic epitopes of the virus, so it can be used for the detection of PCV2 virus. The protein consists of 233 amino acids and has a molecular weight of about 27.2kD.

[0004] Common methods for detecting porcine circovirus type 2 include enzyme-linked immunosorbent assay, colloidal gold immunochromatography, fluorescent immunochromatography, etc. The fluorescent PCR detection method is time-consuming and labor-intensive; colloidal gold and fluorescent immunochromatography are qualitative and semi-quantitative detection methods, and the results are not very accurate. Accurate and rapid detection products are urgently needed, and chemiluminescence is the best choice. Compared with the fluorescent PCR detection method, this technology is simple to operate, has a short reaction time, has good sensitivity and accuracy, and has a wide detection range. Summary of the invention

[0005] In order to solve the above-mentioned problems of the prior art, the present invention provides a porcine circovirus type 2 chemiluminescent detection kit having technical characteristics that can solve the problems of high cost, long time consumption, low accuracy, etc. of PCV2 diagnostic reagents currently on the market.

[0006] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0007] The invention discloses a porcine circovirus type 2 chemiluminescent detection kit, which is characterized in that the kit is provided with a box body, in which a polyclonal antibody coupled with magnetic particles, acridinium ester-labeled monoclonal antibody, porcine circovirus type 2 antigen dry powder, antigen calibration buffer solution, cleaning solution, chemiluminescent pre-excitation solution A, chemiluminescent excitation solution B are contained, and the box body also includes a reaction tube slot and an operation manual slot, in which a plurality of reaction tubes can be detachably placed, and in which a manual is placed.

[0008] Preferably, the heavy chain variable region sequence of the monoclonal antibody is SEQ ID NO.1:

[0009] 1 QYQLEDFSGP GLVASSQSLS ITCTVSGFSL SRYMVHWVRQ PNGKGFSEWL GMIWGGGGSDRYNSALKSRLS

[0010] 71 IGTDNSSQV FLKSEDSLQT DDTAMYYCAR DGYYDYAIDY WGQGTSTVVC SER;

[0011] The heavy chain variable region sequence of the monoclonal antibody is SEQ ID NO.2:

[0012] 1 DIKSQSPSSM YAMLSWERVT ITCASSDICS YLSWYQLKPW KSSKTLDYYA TSADGVPSRFSASGSGQDYY

[0013] 71 LTISSLMSDD TATYYCSE.

[0014] Preferably, the particle size of the magnetic particles is 0.01-1 μm.

[0015] Preferably, the magnetic particle-coupled polyclonal antibody is a magnetic particle directly coupled to a porcine circovirus type 2 polyclonal antibody.

[0016] Preferably, the acridinium ester-labeled monoclonal antibody is directly coupled to a porcine circovirus type 2 monoclonal antibody using acridinium ester.

[0017] Preferably, the antigen standard buffer solution is a phosphate buffer solution containing 0.5-5.0% bovine serum albumin, and a series of calibration solutions with a concentration gradient can be prepared by adding porcine circovirus type 2 antigen dry powder to the phosphate buffer solution.

[0018] Preferably, the chemiluminescent pre-excitation solution A is composed of a mixture of concentrated nitric acid and hydrogen peroxide, wherein the concentration of nitric acid is 0.05-2.5 mol / L and the mass fraction of hydrogen peroxide is 0.05-5%; the chemiluminescent excitation solution B is composed of a mixture of sodium hydroxide and Triton X-100, wherein the concentration of sodium hydroxide is 0.05-1.0 mol / L and the concentration of Triton X-100 is 0.05-2.0 mol / L.

[0019] Preferably, the pH value of the cleaning solution is 7.0-9.0, and the cleaning solution contains a phosphate buffer solution with a concentration of 5.0-50.0 mmol / L and a mass fraction of 0.01-0.25% Tween 20.

[0020] Preferably, the method for preparing the magnetic particle-coupled polyclonal antibody comprises the following steps:

[0021] Step 1) Take 1 mg of carboxyl magnetic particles in a 1.0 mL centrifuge tube, add 0.1-0.5 mL of 0.1 mol / L MES buffer, vortex to mix, place on a magnetic stand, let stand for 5 min to separate the magnetic particles from the liquid, discard the supernatant, wash 3 times, then add 0.5-1.0 mL of MES (pH 6.0) buffer, and vortex;

[0022] Step 2) Add 15 μg of polyclonal antibody, vortex, rotate the reaction tube, and incubate at room temperature for 20 min;

[0023] Step 3) Add 10 μL of 10 mg / mL coupling reagent EDC, vortex, rotate the reaction tube, and incubate at room temperature for 2 h;

[0024] Step 4) After centrifugation, discard the supernatant, add 200 μL of a washing buffer comprising TBS and 0.05% Tween 20, and wash three times;

[0025] Step 5) Blocking is performed with a buffer solution containing 1% bovine serum albumin for 6 hours, and the magnetic particles are resuspended and stored at 2-8°C.

[0026] Preferably, the method for preparing an acridinium ester-labeled monoclonal antibody comprises the following steps:

[0027] Step 1) Weigh 1.6 mg of acridinium ester NSP-DMAE-NHS and dissolve it in anhydrous DMSO to prepare a 6.5 mmol / L mother solution;

[0028] Step 2) In a light-proof environment, 5 μL of monoclonal antibody was mixed with 195 μL of acridinium ester stock solution, reacted at room temperature for 1 h, 100 μL of 10 g / L lysine was added, and the reaction was continued for 10 min to terminate the labeling reaction;

[0029] Step 3) injecting the labeled solution into a Sephadex G-25 desalting column and washing with 0.1 mol / L PBS;

[0030] Step 4) Detecting the protein with a chromatograph, detecting the protein concentration at 280 nm, and collecting the effluent when an obvious protein peak appears on the chromatograph;

[0031] Step 5) 1% bovine serum albumin is added to the effluent and then cryopreserved.

[0032] Preferably, the porcine circovirus type 2 antigen dry powder is mixed with the calibration buffer solution to prepare a standard, and the preparation method comprises the following steps:

[0033] The porcine circovirus type 2 antigen dry powder in the kit was mixed with 1 mL of the calibrator buffer solution to prepare a 1 mg / mL calibrator. The standard was serially diluted to prepare calibrators of 1.0 mg / mL, 100 μg / mL, 10 μg / mL, 1.0 μg / mL, 100 ng / mL, 10 ng / mL, 1.0 ng / mL, and 0 ng / mL, respectively.

[0034] Beneficial effects: The kit of the present invention combines chemiluminescence technology with immunomagnetic particles. Compared with the existing porcine circovirus type 2 detection technology, the kit of the present invention has the advantages of high sensitivity, strong specificity, short reaction time, etc.; the present invention uses genetically expressed recombinant protein as the detection antigen, which has good safety, no foreign protein contamination, and has the advantages of good specificity, high sensitivity, and large-scale detection; the detection time of the present invention is 25 minutes; the reaction tube of the kit is detachable. When the sample is small, the temporarily unused reaction tube can be removed for subsequent use, which can further reduce the use cost and facilitate the use of end users. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a schematic diagram of the structure of the kit of the present invention.

[0036] Figure 2 It is a graph of the luminescence values ​​of the samples detected by the present invention. DETAILED DESCRIPTION

[0037] The following is in conjunction with the instruction manual Figure 1-2 The present invention is further described with reference to the following embodiments, but the present invention is not limited to the following embodiments.

[0038] The invention discloses a porcine circovirus type 2 chemiluminescence detection kit, which is characterized in that the kit is provided with a box body 1, in which a magnetic particle-coupled polyclonal antibody 2, acridinium ester-labeled monoclonal antibody 3, porcine circovirus type 2 antigen dry powder 4, antigen calibration buffer solution 5, cleaning solution 6, chemiluminescence pre-excitation solution A 7, and chemiluminescence excitation solution B 8 are contained, and the box body 1 also comprises a reaction tube slot 9 and an operation manual slot 10, wherein a plurality of reaction tubes are detachably placed in the reaction tube slot 9, and an operation manual slot 10 is placed with a manual, the pH value of the cleaning solution is 7.0-9.0, the cleaning solution contains a phosphate buffer solution with a concentration of 5.0-50.0 mmol / L, and contains Tween 20 with a mass fraction of 0.01-0.25%, and the cleaning solution is used to remove unbound components in the reaction system. The particle size of the magnetic particles is 0.01-1 μm, the polyclonal antibody coupled with the magnetic particles is directly coupled with the porcine circovirus type 2 polyclonal antibody by using the magnetic particles, the monoclonal antibody labeled with acridinium ester is directly coupled with the porcine circovirus type 2 monoclonal antibody by using acridinium ester, the antigen standard buffer solution is a phosphate buffer solution, the phosphate buffer solution contains 0.5-5.0% bovine serum albumin, and a series of calibration solutions with concentration gradients can be prepared by adding porcine circovirus type 2 antigen dry powder to the phosphate buffer solution. The chemiluminescent pre-excitation solution A is composed of a mixture of concentrated nitric acid and hydrogen peroxide, wherein the concentration of nitric acid is 0.05-2.5 mol / L, and the mass fraction of hydrogen peroxide is 0.05-5%, and the chemiluminescent excitation solution B is composed of a mixture of sodium hydroxide and Triton X-100, wherein the concentration of sodium hydroxide is 0.05-1.0 mol / L, and the concentration of Triton X-100 is 0.05-2.0 mol / L.

[0039] Example 1: Preparation of polyclonal antibodies coupled to magnetic particles:

[0040] The polyclonal antibody is prepared by purifying the serum of porcine circovirus inactivated vaccine purchased from Zhejiang Shihua Nuobeiwei Biological after three immunizations of New Zealand white rabbits using a Protein G column.

[0041] 1. Take 1 mg of carboxyl magnetic particles (Shanghai Huizhi) in a 0.5 mL centrifuge tube, add a certain amount of 0.1 mol / L MES buffer, vortex to mix, place on a magnetic rack, let stand for 5 minutes to separate the magnetic particles from the liquid, discard the supernatant, wash 3 times, then add a certain amount of MES pH 6.0 buffer and vortex.

[0042] 2. Add 15 μg of polyclonal antibody, vortex, rotate the reaction tube, and incubate at room temperature for 20 minutes.

[0043] 3. Add 10 μL of 10 mg / mL coupling reagent EDC, vortex, rotate the reaction tube, and incubate at room temperature for 2 hours.

[0044] 4. After centrifugation, discard the supernatant, add 200 μL of washing buffer (TBS + 0.05% Tween 20), and wash 3 times.

[0045] 5. Block with a buffer containing 1% bovine serum albumin for 6 hours. Resuspend the magnetic particles and store at 2-8°C.

[0046] Example 2: Preparation of acridinium ester labeled monoclonal antibody:

[0047] The monoclonal antibody is a mouse anti-PCV2-Cap protein monoclonal antibody, the heavy chain variable region sequence of the monoclonal antibody is shown in SEQ ID NO.1; the light chain variable region sequence of the monoclonal antibody is shown in SEQ ID NO.2.

[0048] 1. Weigh 1.6 mg of acridinium ester NSP-DMAE-NHS (purchased from MacLean) and dissolve it in anhydrous DMSO to prepare a 6.5 mmol / L stock solution.

[0049] 2. In a light-proof environment, mix 5 μL of monoclonal antibody with 195 μL of acridinium ester stock solution, react at room temperature for 1 hour, add 100 μL of 10 g / L lysine, and continue the reaction for 10 minutes to terminate the labeling reaction.

[0050] 3. Inject the labeled solution into a Sephadex G-25 desalting column and wash with 0.1 mol / L PBS.

[0051] 4. Detect protein with a chromatograph and detect protein concentration at 280 nm. When a clear protein peak appears on the chromatograph, collect the effluent.

[0052] 5. Add 1% bovine serum albumin to the effluent and store it in the freezer.

[0053] Example 3: Preparation of Calibrators

[0054] The PCV2-Cap protein prepared from the antigen dry powder in the kit was lyophilized and stored in a -80°C refrigerator. It was mixed with 1 mL of a standard buffer solution containing 2% BSA, 0.5% Tween-20, and 5% trehalose in PBS buffer to prepare a 1 mg / mL calibrator. The calibrator was gradiently diluted to prepare 1.0 mg / mL, 100 μg / mL, 10 μg / mL, 1.0 μg / mL, 100 ng / mL, 10 ng / mL, 1.0 ng / mL, and 0 ng / mL calibrators, respectively.

[0055] Example 4: Kit linear range:

[0056] For 1.0mg / mL, 100μg / mL, 10μg / mL, 1.0μg / mL, 100ng / mL, 10ng / mL, 1.0ng / mL, 0ng / mL calibrants. Figure 2 The standard curve after fitting is shown in Figure 2. 2 The kit has good linearity in the concentration range of 0.25ng / mL-1.0mg / mL.

[0057] Example 5: Kit detection limit:

[0058] The zero standard solution was tested 20 times, and the average value of the zero standard solution plus 3 times the standard deviation was taken as the detection limit of this kit. The detection limit of this kit is 0.051 ng / mL. As shown in Table 1.

[0059] Table 1 Detection limits

[0060]

[0061] Comparison with fluorescent PCR detection method:

[0062] 70 serum samples were tested using the kit of the present invention and a commercially available fluorescent PCR kit, with each sample tested 3 times. Using the results of fluorescent PCR as the standard, the results of 68 samples tested by chemiluminescence were the same as those of PCR, with a sample compliance rate of 97.14%.

[0063] Table 2 Comparative analysis between chemiluminescence detection and PCR

[0064]

[0065] Finally, it should be noted that the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A chemiluminescent detection kit for porcine circovirus type 2, characterized in that: The kit is provided with a box body, which contains magnetic particle-coupled polyclonal antibodies, acridinium ester-labeled monoclonal antibodies, porcine circovirus type 2 antigen dry powder, antigen calibration buffer solution, cleaning solution, chemiluminescent pre-excitation solution A, chemiluminescent excitation solution B, and also includes a reaction tube slot and an operation manual slot, wherein a plurality of reaction tubes can be detachably placed in the reaction tube slot, and an instruction manual is placed in the operation manual slot; The monoclonal antibody is a mouse anti-PCV2-Cap protein monoclonal antibody, the heavy chain variable region sequence of the monoclonal antibody is shown in SEQ ID NO.1; the light chain variable region sequence of the monoclonal antibody is shown in SEQ ID NO.2; The preparation method of the acridinium ester-labeled monoclonal antibody: Step 1: Weigh 1.6 mg of acridinium ester NSP-DMAE-NHS and dissolve it in anhydrous DMSO to prepare a 6.5 mmol / L stock solution; Step 2: In a light-proof environment, 5 μL of the monoclonal antibody was mixed with 195 μL of acridinium ester stock solution, and the mixture was reacted at room temperature for 1 h. 100 μL of 10 g / L lysine was added, and the reaction was continued for 10 min to terminate the labeling reaction. Step 3: Inject the labeled solution into a Sephadex G-25 desalting column and wash with 0.1 mol / L PBS; Step 4: Detect the protein using a chromatograph and detect the protein concentration at 280 nm. When an obvious protein peak appears on the chromatograph, collect the effluent; Step 5: Add 1% bovine serum albumin to the effluent and store it in the freezer.

2. A porcine circovirus type 2 chemiluminescent detection kit according to claim 1, characterized in that, The particle size of the magnetic particles is 0.01-1 μm.

3. A porcine circovirus type 2 chemiluminescent detection kit according to claim 1 or 2, characterized in that: The magnetic particle-coupled polyclonal antibody is directly coupled with the porcine circovirus type 2 polyclonal antibody by using the magnetic particle.

4. A porcine circovirus type 2 chemiluminescent detection kit according to claim 1, characterized in that: The acridinium ester-labeled monoclonal antibody is directly coupled with porcine circovirus type 2 monoclonal antibody using acridinium ester.

5. A porcine circovirus type 2 chemiluminescent detection kit according to claim 1, characterized in that: The antigen calibration buffer solution is a phosphate buffer solution containing 0.5-5.0% bovine serum albumin. A series of calibration solutions with concentration gradients can be prepared by adding porcine circovirus type 2 antigen dry powder to the phosphate buffer solution.

6. A chemiluminescent detection kit for porcine circovirus type 2 according to claim 1, 2, 4 or 5, characterized in that: The chemiluminescent pre-excitation solution A is composed of a mixture of concentrated nitric acid and hydrogen peroxide, wherein the concentration of nitric acid is 0.05-2.5 mol / L and the mass fraction of hydrogen peroxide is 0.05-5%. The chemiluminescent excitation solution B is composed of a mixture of sodium hydroxide and Triton X-100, wherein the concentration of sodium hydroxide is 0.05-1.0 mol / L and the concentration of Triton X-100 is 0.05-2.0 mol / L.

7. A chemiluminescent detection kit for porcine circovirus type 2 according to claim 1, characterized in that: The pH value of the cleaning solution is 7.0-9.0, and the cleaning solution contains a phosphate buffer solution with a concentration of 5.0-50.0 mmol / L and Tween 20 with a mass fraction of 0.01-0.25%.

8. A chemiluminescent detection kit for porcine circovirus type 2 according to claim 1, characterized in that: The method for preparing the magnetic particle-coupled polyclonal antibody comprises the following steps: Step 1: Take 1 mg of carboxyl magnetic particles in a 1.0 mL centrifuge tube, add 0.1-0.5 mL of 0.1 mol / L MES buffer, vortex to mix, place on a magnetic stand, let stand for 5 min to separate the magnetic particles from the liquid, discard the supernatant, wash 3 times, then add 0.5-1.0 mL of MES buffer with a pH value of 6.0, and vortex; Step 2: Add 15 μg of polyclonal antibody, vortex, rotate the reaction tube, and incubate at room temperature for 20 min; Step 3: Add 10 μL of 10 mg / mL coupling reagent EDC, vortex, rotate the reaction tube, and incubate at room temperature for 2 h; Step 4: After centrifugation, discard the supernatant, add 200 μL of a washing buffer comprising TBS and 0.05% Tween 20, and wash three times; Step 5: Block with a buffer containing 1% bovine serum albumin for 6 h, resuspend the magnetic particles and store at 2-8°C.

9. A chemiluminescent detection kit for porcine circovirus type 2 according to claim 1, characterized in that: The porcine circovirus type 2 antigen dry powder is mixed with the calibration buffer solution to prepare a calibration product, and the preparation method comprises the following steps: The porcine circovirus type 2 antigen dry powder in the kit was mixed with 1 mL of the calibrator buffer solution to prepare a 1 mg / mL calibrator. The calibrator was serially diluted to prepare 1.0 mg / mL, 100 μg / mL, 10 μg / mL, 1.0 μg / mL, 100 ng / mL, 10 ng / mL, 1.0 ng / mL, and 0 ng / mL calibrators, respectively.

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