Rapid detection method for PRRSV N antibody particle control chip

Abstract

The invention is suitable for the field of animal epidemic disease diagnosis, and provides a PRRSV N antibody particle control chip rapid detection method, which comprises the following steps: S1, diluting purified PRRSV N protein with a PBS buffer solution, coating a pretreated chip with the diluted PRRSV N protein, and incubating at 40 L / hole and 4 DEG C overnight; s2, washing with 0.1% PRST for three times, patting to be dry, adding 100L / hole confining liquid, and incubating for 1h under the condition of normal temperature; s3, not washing, adding 40 L of diluted serum to be detected per hole, and incubating in an incubator for 30-2 minutes; s4, washing with 0.05% PBST for 4 times, patting to be dry, adding diluted goat anti-pig HRP 40L / hole, incubating in an incubator for 30 seconds to 2 minutes, and then selectively accelerating for 30 seconds to 1 minute again; s5, washing with 0.05% PBST for 4 times, adding a chemiluminescence substrate of 40L / hole, reacting at room temperature in a dark place for 0-3 minutes, and measuring a chemiluminescence value at the wavelength of 425nm by using a full-automatic chemiluminescence detector; the method has the advantages of being rapid in detection, high in accuracy and wide in applicability.

Description

Technical Field

[0001] The present invention belongs to the field of animal disease diagnosis, and in particular relates to a rapid detection method for a PRRSV N antibody microparticle manipulation chip. Background Art

[0002] Animal diseases severely hamper the healthy breeding of livestock and poultry, and accurate and timely diagnosis is crucial for disease prevention and control. Current diagnostic technologies lack speed, specificity, and sensitivity, failing to meet practical needs. Traditional enzyme-linked immunosorbent assays (ELISAs), a commonly used method for animal disease diagnosis, rely on vibrational diffusion, thermal diffusion, and molecular Brownian motion for antigen-antibody binding. The incubation period, which takes 30 minutes to an hour, is inefficient and time-consuming. Furthermore, nonspecific reactions due to physical adsorption can affect diagnostic speed, specificity, and sensitivity.

[0003] Current PRRSV antibody detection methods, such as the enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), immunoperoxidase monolayer assay (IPMA), colloidal gold immunochromatographic assay (GICA), and latex agglutination test (LAT), all have limitations. IPMA, IFA, and LAT are limited by the relevance of the experimental strain to the field strain, operator expertise, and laboratory location. ELISA is not suitable for on-site testing and is time-consuming. GICA has low sensitivity. Therefore, a rapid PRRSV N antibody microparticle-manipulated chip-based detection method is needed to address these issues. Summary of the Invention

[0004] The purpose of the embodiments of the present invention is to provide a method for rapid detection of PRRSV N antibody microparticles by a chip manipulation to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions: The rapid detection method of PRRSV N antibody microparticle manipulation chip includes the following steps: S1. Dilute the purified PRRSV N protein with PBS buffer and coat it on the pretreated chip at 40 µL / well. Incubate at 4°C overnight. S2, wash three times with 0.1% PRST, pat dry, add 100 μL / well of blocking solution, and incubate at room temperature for 1 hour; S3: Without washing, add 40 µL / well of the diluted serum to be tested and incubate in the incubator for 30 seconds to 2 minutes. S4. Wash four times with 0.05% PBST, pat dry, add 40 µL / well of diluted goat anti-swine HRP (secondary antibody), and incubate in an incubator for 30 seconds to 2 minutes. You can then accelerate again for 30 seconds to 1 minute. S5. Wash four times with 0.05% PBST, add 40 µL / well of chemiluminescent substrate, react at room temperature in the dark for 0-3 minutes, and then use an automatic chemiluminescence detector to measure the chemiluminescence value at a wavelength of 425 nm.

[0006] In a further technical solution, the optimal concentration of the N protein for coating is 10 μg / mL.

[0007] In a further technical solution, the blocking solution is 10% BSA.

[0008] According to a further technical solution, the accelerating voltage of the micro-immunoaccelerator is 6V, the accelerating frequency is 20K, and the accelerating time N protein is 1 min.

[0009] In a further technical solution, the goat anti-pig HRP (secondary antibody) is screened at a concentration that maximizes the P / N value.

[0010] According to a further technical solution, the optimal dilution of the serum to be tested is 1:40.

[0011] Compared with the prior art, the present invention has the following beneficial effects: This invention offers rapid detection: Based on microparticle manipulation (MMC) technology, it utilizes electrokinetic effects such as alternating current heating to actively control the movement of the target to be tested, accelerating antigen-antibody binding and completing the reaction within seconds. The entire test can be completed within 3-5 minutes at room temperature, significantly shortening the detection time and increasing the efficiency by 60 to 120 times compared to traditional ELISA. The present invention has high accuracy: by optimizing the antigen coating method of the diagnostic chip, the optimal coating solution concentration and time, the optimal blocking solution type and blocking time, the incubation voltage, frequency, secondary antibody concentration, and accelerated incubation time, the established detection method has good sensitivity and specificity, and a high consistency rate compared with commercial kits, with a kappa value of up to 0.925; The present invention has wide applicability: the equipment used is portable and suitable for multiple applications in laboratories, fields, and other scenarios. It is suitable for large-scale PRRSV antibody monitoring, can save manpower, material resources and financial resources, and provide strong support for the prevention and control of disease transmission, daily monitoring, and the formulation of follow-up plans for disease quarantine.

[0012] In order to more clearly illustrate the structural features and effects of the present invention, the present invention is described in detail below with reference to the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a flow chart of the PRRSV microparticle manipulation antibody detection process of the present invention; Figure 2 Graph showing determination of optimal antigen coating concentration and serum dilution for MMC of the present invention; Figure 3 Schematic diagram of screening BN protein for the best coating solution type of the present invention; Figure 4 Schematic diagram of the optimized BN protein of the blocking solution of the present invention; Figure 5 This is a graph of voltage, frequency and time of CD N protein for screening the optimal conditions of the incubator of the present invention; Figure 6 Schematic diagram of screening the optimal goat anti-swine HRP concentration of BN protein in the present invention; Figure 7 Schematic diagram of the specificity determination of MMC of the present invention; Figure 8 This is a specificity test table for the MMC N antibody detection of the present invention; Figure 9 This is a sensitivity test table for the MMC antibody detection method of the present invention; Figure 10 This is a repeatability test table of the MMC N protein detection method of the present invention; Figure 11 This is a comparison table of the test results of the MMC N protein of the present invention and the kit. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0015] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0016] Example 1 Reagent configuration: 5% BSA: Dissolve 0.5 g BSA in 10 mL PBST and mix thoroughly. 10% BSA: Dissolve 1g BSA in 10mL PBST and mix thoroughly; 10% goat serum: Add 10 ml of goat serum to 90 ml of prepared mLPBST and mix thoroughly; 10% rabbit serum: add 10 ml of rabbit serum to 90 ml of prepared mLPBST and mix thoroughly; 10% horse serum: Add 10 ml of horse serum to 90 ml of prepared mLPBST and mix thoroughly; 10% bovine serum: Add 10 ml of bovine serum to 90 ml of prepared mLPBST and mix thoroughly; Chemiluminescent solution: First, equilibrate the reagent to room temperature; then mix substrate A and substrate B in a 1:1 ratio before use; TB buffer: Weigh 4.84 g Trisbase, 0.44 g CaCl2, and 0.19 g MgCl2 into a 2 L glass bottle. Add 1600 mL of 0.85% saline to the bottle, shake well, adjust the pH to 7.6, and fill up to 2 L with saline. Carbonate buffer (CBS): Weigh 2.93 g NaHCO3 and 1.59 g Na2CO3, add them to a 1 L brown glass bottle, then add 800 mL of ultrapure water, mix well, adjust the pH to 9.6, and fill with ultrapure water to 1 L. Phosphate buffered saline (PBS): Weigh 8 g NaCl, 0.2 g KCl, 40.2 g KH2PO4, and 2.9 g Na2HPO4·12H2O and add them to a 1 L brown glass bottle. Add 800 mL of ultrapure water to the brown glass bottle, mix well, adjust the pH to 7.4, and fill up to 1 L with ultrapure water. 0.05% PBST solution: Pipette 1 ml of Tween-20 and add it to 1999 ml of prepared PBS and mix thoroughly; like Figures 1-11 As shown, the embodiment of the present invention provides a method for rapid detection of PRRSV N antibody microparticle manipulation chip, comprising the following steps: S1. Dilute the purified N protein to 10 μg / mL with PBS buffer, coat 40 μL / well on the pretreated chip, and incubate at 4°C overnight. The pre-treated chip surface has been specially chemically modified to increase the protein adsorption capacity while reducing non-specific adsorption.

[0017] S2, wash three times with 0.1% PRST, pat dry, add 100 μL / well of 10% BSA blocking solution, and incubate at room temperature for 1 hour; The function of the blocking solution is to block the uncoated sites on the chip surface and reduce nonspecific binding.

[0018] S3: Without washing, add 40 μL / well of the serum to be tested diluted 1:40 and incubate in a micro-immunoaccelerator with the accelerating voltage set to 6 V, the accelerating frequency set to 20 K, and the accelerating time set to 1 min. The micro-immunoaccelerator accelerates the binding of PRRSV N antibodies in serum to antigens on the chip through the micro-electric field generated by the microelectrode array.

[0019] S4. Wash four times with 0.05% PBST, pat dry, add 40 µL / well of the optimal dilution of goat anti-swine HRP determined through screening, and incubate again in a micro-immunoaccelerator for 30 seconds; Accelerating the reaction again can further improve the binding efficiency of the secondary antibody to the bound antibody.

[0020] S5. Wash the plate four times with 0.05% PBST, add 40 µL / well of luminol chemiluminescent substrate, react at room temperature in the dark for 1 min, and then use an automatic chemiluminescence detector to measure the chemiluminescence value at a wavelength of 425 nm.

[0021] The chemiluminescent substrate reacts under the action of the enzyme, and the intensity of the light signal generated is proportional to the content of PRRSV N antibodies in the serum.

[0022] Example 2 The difference between this embodiment and embodiment 1 is that it includes the following steps: S4. Wash four times with 0.05% PBST, pat dry, add 40 µL / well of the optimal dilution of goat anti-swine HRP determined through screening, and incubate again in a micro-immunoaccelerator for 1 min. Compared with Example 1, the goat anti-porcine HRP (secondary antibody) can bind more fully to the bound antibody, thereby improving the sensitivity of the detection, because the longer accelerated incubation time can increase the chance of the secondary antibody binding to the antibody, allowing more secondary antibodies to bind to the bound antibody, thereby generating a stronger signal in the subsequent chemiluminescence detection; S5. Wash four times with 0.05% PBST, add 40 µL / well of luminol chemiluminescent substrate, react at room temperature in the dark for 3 minutes, and then measure the chemiluminescence value at a wavelength of 425 nm using an automatic chemiluminescence detector; Extending the chemiluminescent substrate reaction time to 3 minutes may make the chemiluminescent reaction more complete and further enhance the detection signal. A longer reaction time allows more chemiluminescent substrates to react under the action of the enzyme, generating more light signals, thereby improving the accuracy and repeatability of the detection. However, excessively long reaction times may also lead to an increase in background signals, so optimization is required in practical applications.

[0023] Example 3 The difference between this embodiment and embodiment 2 is that it includes the following steps: S4. Wash four times with 0.05% PBST, pat dry, add 40 µL / well of the optimal dilution of goat anti-swine HRP determined through screening, and incubate again in a micro-immunoaccelerator for 40 seconds; The accelerated incubation time was 40 seconds, which is between that in Example 1 and Example 2. This time may be used to further explore the optimal accelerated incubation time to balance the sensitivity and efficiency of the test. A shorter accelerated incubation time may reduce the occurrence of nonspecific binding and improve the specificity of the test, but it may also reduce the sensitivity. By adjusting this time, an optimal balance can be found to make the test results more accurate and reliable. S5. Wash four times with 0.05% PBST, add 40 µL / well of luminol chemiluminescent substrate, react at room temperature in the dark for 2 minutes, and then measure the chemiluminescence value at a wavelength of 425 nm using an automatic chemiluminescence detector; The chemiluminescent substrate reaction time is 2 minutes, and we are also exploring the optimal reaction time. A 2-minute reaction time may reduce the interference of background signals while ensuring the detection signal intensity, thereby improving the accuracy of detection. Different reaction times will affect the reaction degree of the chemiluminescent substrate and the generation of light signals, so experiments are needed to determine the optimal reaction time.

[0024] The working principle of this invention is: Based on microparticle manipulation (MMC) technology, it uses the electrokinetic effect (micro electric field) such as AC electrothermal to actively control the movement of the PRRSV N antibody (antigen / antibody) to be tested, accelerating its binding with the antigen coated on the chip to form a specific immune response; The specific process is as follows: first, the purified PRRSV N protein is coated on the chip. After incubation, washing, and blocking, the serum to be tested is added. The PRRSV N antibodies in the serum quickly bind to the antigen on the chip under the action of a micro-electric field. Then, an enzyme-labeled secondary antibody (goat anti-porcine HRP) is added, and the micro-electric field also accelerates the binding of the bound antibody. Finally, a chemiluminescent substrate is added, and the presence and content of PRRSV N antibodies in the serum are determined by measuring the chemiluminescence value. By optimizing the conditions of each step, including antigen and antibody concentration, blocking solution type, incubation conditions, etc., the rapidity, specificity and sensitivity of the detection are ensured.

[0025] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A rapid detection method for PRRSV N antibody microparticle manipulation chip, characterized in that: The following steps are involved: S1. Dilute the purified PRRSV N protein with PBS buffer and coat it on the pretreated chip at 40 µL / well. Incubate at 4°C overnight. S2, wash three times with 0.1% PRST, pat dry, add 100 μL / well of blocking solution, and incubate at room temperature for 1 hour; S3: Without washing, add 40 µL / well of the diluted serum to be tested and incubate in the incubator for 30 seconds to 2 minutes. S4. Wash four times with 0.05% PBST, pat dry, add 40 µL / well of diluted goat anti-pig HRP, and incubate in an incubator for 30 seconds to 2 minutes. You can then accelerate again for 30 seconds to 1 minute. S5. Wash four times with 0.05% PBST, add 40 µL / well of chemiluminescent substrate, react at room temperature in the dark for 0-3 minutes, and then use an automatic chemiluminescence detector to measure the chemiluminescence value at a wavelength of 425 nm.

2. The PRRSV N antibody microparticle manipulation chip rapid detection method according to claim 1, characterized in that: The optimal concentration of the N protein for coating was 10 μg / mL.

3. The PRRSV N antibody microparticle manipulation chip rapid detection method according to claim 1, characterized in that: The blocking solution is 10% BSA.

4. The PRRSV N antibody microparticle manipulation chip rapid detection method according to claim 1, characterized in that: The micro-immunoaccelerator has an accelerating voltage of 6V, an accelerating frequency of 20K, and an accelerating time of 1 min.

5. The PRRSV N antibody microparticle manipulation chip rapid detection method according to claim 1, characterized in that: The goat anti-pig HRP is screened at a concentration that maximizes the P / N value.

6. The method for rapid detection of PRRSV N antibody microparticles by chip manipulation according to claim 1, characterized in that: The optimal dilution of the serum to be tested is 1:40.

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