Method for quantitatively detecting PRRS (porcine reproductive and respiratory syndrome) virus particles and application thereof

Abstract

The invention belongs to the technical field of PRRS (porcine reproductive and respiratory syndrome) virus particle antigen quantitative analysis, and particularly discloses a method for quantitatively detecting PRRS virus particles and application of the method. According to the method, a sucrose density gradient ultracentrifugation method is combined with a high performance liquid chromatograph to quantitatively detect the PRRS virus particles; comprising the following steps: firstly, preparing a 15-45% sucrose gradient by using an automatic gradient mixer, slowly adding a PRRS virus liquid sample at the top of the sucrose gradient, and horizontally and centrifugally separating PRRS virus particles; connecting the sucrose gradient solution with a pumping system of a high performance liquid chromatograph, continuously detecting optical signals of the sucrose gradient solution through an ultraviolet and laser detector, and integrating a chromatographic peak to obtain a peak area; and calculating the content of the PRRS virus particles according to the regression equation of the ultraviolet absorption peak area and the protein concentration of the thyroglobulin standard substance. The method has better accuracy, repeatability and stability, and is suitable for measuring the antigen content of the PRRS virus particles.

Description

Technical Field

[0001] This invention patent belongs to the field of PRRS virus particle antigen quantitative analysis technology, specifically relating to a method for quantitative detection of PRRS virus particles and its application. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease characterized by abortion in pregnant sows and respiratory disorders in piglets, and it is prone to large-scale outbreaks. The pathogen of PRRS is the porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped RNA virus. PRRSV has two genotypes: European (type I) and American (type II). It exhibits immunosuppressive and persistent infection characteristics and is prone to mutation and recombination.

[0003] Vaccination is an effective way to control PRRS outbreaks. Currently, the most clinically mature PRRS vaccines both domestically and internationally are inactivated vaccines and live attenuated vaccines, but these two types of vaccines still have potential safety and efficacy concerns. Therefore, improving vaccine manufacturing processes and strictly controlling vaccine quality are crucial. The key to PRRS vaccine industrial production and quality assessment lies in the quantitative technology of PRRS viral particles. Accurate quantification of PRRS viral particles during vaccine production can guide improvements in vaccine manufacturing processes, thereby increasing production efficiency and saving costs; the viral particle content in the vaccine directly affects the final immune protective effect and safety. Therefore, establishing a relatively accurate and simple method for detecting PRRS viral particle content has significant application value.

[0004] Currently, the detection of PRRS viral load in bodily fluids generally uses the TCID (Cellular Half-Infectious Dose). 50 While real-time quantitative PCR and other methods exist, the sensitivity, accuracy, and repeatability of both are difficult to guarantee. In particular, a quantitative method for PRRS inactivated virus solution in industrial production remains lacking, making it impossible to accurately determine the final immunization dose of the vaccine, thus leading to significant side effects from inactivated vaccines. Sucrose density gradient centrifugation is a classic method for virus isolation; sucrose is relatively mild and helps maintain the integrity of virus particles. However, traditional sucrose density gradient centrifugation is entirely manual, cumbersome, has poor repeatability, is time-consuming, and incurs high labor and operating costs, significantly reducing the accuracy of quantifying PRRSV using this method.

[0005] Therefore, this paper proposes a method for quantitative detection of PRRS virus particles and its application. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides a method for quantitative detection of PRRS virus particles and its application. In this invention, a sucrose density gradient is prepared using a fully automated gradient analyzer, and a high-performance liquid chromatography (HPLC) system is used to achieve fully automated peak analysis of PRRS virus particles. A standard curve is established using standard proteins of known concentrations, and the results for the test sample are calculated. This method overcomes the problems of poor repeatability and complex operation of traditional sucrose density gradient ultracentrifugation methods, and has advantages such as short detection cycle, high sensitivity, good repeatability, and small batch-to-batch error.

[0007] To achieve the above-mentioned technical effects, the first objective of this invention is to provide a method for quantitative detection of PRRS virus particles, specifically including the following steps: S1. Preparation of sucrose gradient: Prepare 15% and 45% sucrose solutions using TN buffer (pH 8.0); mark 12.5ml ultracentrifuge tubes using the scale on the gradient mixer; first add 15% sucrose solution to the marked position on the ultracentrifuge tube, then slowly add 45% sucrose solution from the bottom to the top of the ultracentrifuge tube using a long needle; then place it on the gradient mixer, select the 15%-45% sucrose gradient preparation program, and run continuously for 113s to obtain a 15%-45% continuous gradient sucrose solution; S2, Sucrose density gradient ultracentrifugation: The PRRS virus particle sample was centrifuged at 8000 rpm for 3 min at 4℃, and the supernatant was collected for later use; 1.0 ml of sucrose continuous gradient solution was discarded, and then 1.5 ml of supernatant was slowly added to the top layer. The sample was centrifuged horizontally at 35000 rpm at 10℃ for 3.5 h to obtain the centrifuged sample. S3. Preparation of the high performance liquid chromatograph: Connect the pump head of the high performance liquid chromatograph to the external mobile phase (45% sucrose solution) with a tubing with a 15cm needle tip and a diameter of 0.5mm. Turn on the pumping system and ultraviolet detection system of the high performance liquid chromatograph and balance the baseline. S4. Detection of PRRS virus particle content: Insert the long needle from S3 into the bottom of the ultracentrifuge tube containing the centrifuged sample; set the flow rate to 1.0 ml / min and the detection wavelength to 280 nm. The computer system of the chromatograph will automatically draw the chromatogram, and manual integration will be performed to obtain the target peak area. S5. Prepare six concentration gradient standard solutions of thyroglobulin standard protein in the range of 10-160 µg / ml using TN buffer (pH 8.0). Take 1.5 ml of the standard solution sample and follow the steps in S1-S3. After equilibrating the baseline, insert a long needle into the bottom of the ultracentrifuge tube containing the standard solution sample. Run the centrifuge at a flow rate of 1.0 ml / min and detect at a UV wavelength of 280 nm. Integrate the absorption peaks in the chromatogram to obtain the peak area and establish a linear regression equation between the absorption peak area and the protein concentration. S6. Based on the linear regression equation between protein concentration and peak area established in S5, substitute the target absorption peak area of ​​PRRS virus particles into the regression equation to calculate the content of PRRS virus particles in the sample to be tested.

[0008] Furthermore, in S1, before using the gradient mixer, the gradient mixer should be adjusted to balance using a level.

[0009] Furthermore, in S3, deionized water is used as the mobile phase to remove air bubbles from the system.

[0010] A second objective of this invention is to provide the application of the above-described method for quantitative detection of PRRS virus particles in evaluating the antigen content of PRRS vaccines.

[0011] A third objective of this invention is to provide the application of the above-described method for quantitative detection of PRRS virus particles in the preparation of PRRS vaccines.

[0012] The beneficial effects of this invention are: The sucrose density gradient ultracentrifugation method in this invention operates under mild conditions, causing minimal damage to virus particles. The optimized sucrose gradient and centrifugation conditions result in more distinct sucrose density gradient layers. The combined use of high-performance liquid chromatography (HPLC) avoids the cumbersome nature of manual operation and the subjectivity of human judgment, thereby improving the sensitivity, repeatability, accuracy, and efficiency of the sucrose density gradient ultracentrifugation quantitative method. Compared with conventional methods, this method is simple, practical, rapid, and sensitive, suitable for determining the antigen content of PRRS virus particles, and can be used to guide PRRS vaccine antigen production and quality assessment. Attached Figure Description

[0013] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0014] Figure 1 Chromatographic analysis of PRRS virus particle samples obtained by sucrose density gradient ultracentrifugation combined with high performance liquid chromatography; Figure 2The image shows the transmission electron microscopy analysis results of the target peak collected by high performance liquid chromatography; Figure 3 This is a chromatogram of the sucrose density gradient ultracentrifugation analysis of the thyroglobulin standard protein in Example 1; Figure 4 This is a linear relationship between the absorption peak area and concentration of thyroglobulin at different dilution concentrations in Example 1. Figure 5 The image shows the chromatographic results of sucrose density gradient ultracentrifugation after three PRRS virus particle samples in Example 2. Detailed Implementation

[0015] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Example 1

[0016] The existing technology in this embodiment has the following problems: because the sucrose density gradient centrifugation method is a classic method for separating viruses at the present stage, sucrose is relatively mild and is conducive to maintaining the integrity of virus particles. However, the traditional sucrose density gradient centrifugation method is all done manually, which is cumbersome, has poor repeatability, long cycle, and high labor and operating costs, which greatly reduces the accuracy of quantifying PRRSV by sucrose density gradient centrifugation.

[0017] Therefore, the inventors provided that the primary objective of this invention is to provide a method for quantifying PRRS virus particles, and the determination method specifically includes the following steps; S1, Preparation of the sucrose gradient: (1) Prepare 15% and 45% sucrose solutions using TN buffer (pH 8.0); (2) Take a 12.5ml ultracentrifuge tube and mark it with the scale on the gradient mixer. (3) First, add 15% sucrose solution to the ultracentrifuge tube up to the mark, then use a long needle to add 45% sucrose solution from the bottom to the top of the ultracentrifuge tube. No air bubbles should be generated during the whole process. (4) Turn on the gradient mixer, use a level to adjust the gradient mixer to equilibrium, place the ultracentrifuge tube containing the sucrose gradient solution into the gradient mixer, select the 15%-45% sucrose gradient preparation program, and work continuously for 113s to obtain a 15%-45% continuous gradient sucrose solution.

[0018] S2, sucrose density gradient ultracentrifugation: (1) Take a PRRS virus particle sample, centrifuge at 8000 rpm for 3 min at 4℃, and take the supernatant for later use; (2) Discard 1.0 ml of the prepared sucrose gradient solution, add 1.5 ml of supernatant, and centrifuge horizontally at 10℃ and 35000 rpm for 3.5 h to obtain the centrifuged sample; S3. Preparation of the high-performance liquid chromatograph: (1) Connect the pump head of the high performance liquid chromatograph to the external mobile phase using a tubing with a 15cm long needle tip and a diameter of 0.5mm. (2) Turn on the pumping system and ultraviolet detection system of the high performance liquid chromatograph, and use deionized water as the mobile phase to remove air bubbles in the system; (3) Use a 45% sucrose solution as the mobile phase to equilibrate the baseline.

[0019] Detection of S4 and PRRS virus particles: (1) Insert the long needle from step 3 into the bottom of the ultracentrifuge tube from step 2; (2) Turn on the pump system, set the flow rate to 1.0 ml / min, set the detection wavelength to 280 nm, the computer system of the chromatograph will automatically draw the chromatogram, perform manual integration, and obtain the target peak area; The chromatograms plotted from the measured data are as follows: Figure 1 As shown, the chromatographic peak position of the PRRS virus particle sample is 5-7 min (Figure A).

[0020] When performing chromatograph analysis, open the fraction collector, collect the target peak position, and store it in the collection tube at 2–8°C for later use.

[0021] Transmission electron microscopy (TEM) of the target peak samples collected in the above steps reveals complete PRRS virus particles, such as... Figure 2 As shown.

[0022] The target peak samples collected in the above steps were subjected to real-time PCR detection, and PRRSV was detected, as shown in Table 1. Table 1 Sample Name Cq value Positive control 15.01 negative control N / A Target peak sample 15.40 S5. Prepare six concentration gradient standard solutions of thyroglobulin standard protein in the range of 10-160 µg / ml using TN buffer (pH 8.0). Take 1.5 ml of the standard solution sample and follow the steps in S1-S3. After equilibrating the baseline, insert a long needle into the bottom of the ultracentrifuge tube containing the standard solution sample. Run the centrifuge at a flow rate of 1.0 ml / min and detect at a UV wavelength of 280 nm. Integrate the absorption peaks in the chromatogram to obtain the peak area and establish a linear regression equation between the absorption peak area and the protein concentration. S6. Based on the linear regression equation between protein concentration and peak area established in S5, substitute the target absorption peak area of ​​PRRS virus particles into the regression equation to calculate the content of PRRS virus particles in the sample to be tested.

[0023] In this embodiment, based on S1, the gradient mixer should be adjusted to balance using a level before use.

[0024] In this embodiment, based on S3, deionized water is also used as the mobile phase to remove air bubbles from the system.

[0025] Based on the above steps, the present invention provides a preferred list of the equipment and materials required for the experiment, but the above list does not exclude the use of other equipment and materials with the same function, as detailed below; (1) Instruments and equipment The ultra-fast refrigerated centrifuge was purchased from Beckman Coulter's Optima XE-90 model; the high-speed centrifuge was purchased from Shuke Coulter's TGL-16 model; the high-performance liquid chromatograph was purchased from Shimadzu's LC-40 model; and the fully automated density gradient preparation system was purchased from Biocomp Coulter.

[0026] (2) Reagents Thyroglobulin was purchased from GE; NaCl was purchased from Sinopharm Chemical Reagent Co., Ltd.; chromatographic grade methanol and isopropanol were purchased from Sigma-Aldrich; Tris was purchased from VWR Life Science; sucrose was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0027] (3) PRRS virus fluid sample The PRRS virus solution is prepared and preserved by the R&D center of Huapai Biotechnology Group. No treatment is required for the PRRS virus solution during sample testing, and it can be directly measured. Example 2

[0028] Based on the above embodiments, this embodiment provides the application of a method for quantitative detection of PRRS virus particles in evaluating the antigen content of PRRS vaccines. Example 3

[0029] Based on the above embodiments, this embodiment provides the application of a method for quantitative detection of PRRS virus particles in the preparation of PRRS vaccines. Example 4

[0030] To verify the high accuracy of this method, the present invention also uses thyroglobulin standard protein for validation; Prepare six concentration gradient standard solutions of thyroglobulin using TN buffer (pH 8.0) at concentrations ranging from 10 to 160 µg / ml, and store at 2–8°C for later use. Following the steps of step 1 (preparation of sucrose gradient), step 2 (ultracentrifugation of sucrose density gradient), step 3 (preparation of high performance liquid chromatography), and step 4 (determination of PRRS virus particle content), the virus sample was replaced with thyroglobulin standard protein, and the concentration of the standard protein was analyzed by chromatography. like Figure 3 As shown, the standard thyroglobulin protein exhibits a single chromatographic absorption peak. The concentrations of the standard solutions diluted in the experiment were 0 µg / ml, 10 µg / ml, 20 µg / ml, 40 µg / ml, 80 µg / ml, and 160 µg / ml, respectively.

[0031] By integrating the absorption peaks of standard proteins at different concentrations, the peak areas are obtained. With the peak area as the abscissa and the protein content as the ordinate, a linear response trend line is obtained, which yields C = (4E-05X+2.3781) / 1.5. Where C is the protein concentration in µg / ml and X is the absorption peak area in mV·min, see [reference needed]. Figure 4 The R value of the obtained standard curve 2 The value is 0.9989, indicating that this method has high accuracy. Example 5

[0032] Establishment and repeatability of a quantitative method for PRRS virus particle samples; 1. Integration of absorption peaks of PRRS virus particle samples: After detecting three PRRS virus particle samples according to the assay method, the target peaks were integrated; the assay was repeated three times. 2. Calculation of PRRS virus particle content in sample: Substitute the target absorption peak area into the formula C=(4E-05X+2.3781) / 1.5 obtained in Example 1 to calculate the antigen concentration, where C is the sample protein concentration in µg / ml and X is the absorption peak area in mV.min. Each sample is measured three times.

[0033] The results of sucrose density gradient centrifugation combined with high performance liquid chromatography analysis are shown below. Figure 5 Figures A, B, and C are chromatograms of three batches of samples, and the results are shown in Table 2. Table 2 The PRRS virus particle content was repeatedly detected by sucrose density gradient centrifugation combined with high performance liquid chromatography (HPLC). The peak positions of the PRRS virus particles were consistent. The antigen content statistics in Table 2 prove that the detection of PRRS virus particle content by sucrose density gradient centrifugation combined with HPLC is reproducible.

[0034] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0035] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for quantitatively detecting PRRS virus particles, characterized by, Specifically, the following steps are included: S1. Preparation of sucrose gradient: Prepare 15% and 45% sucrose solutions respectively using TN buffer (pH 8.0); Mark the 12.5ml ultracentrifuge tubes using the scale on the gradient mixer; first add 15% sucrose solution to the marked position on the ultracentrifuge tube, then slowly add 45% sucrose solution from the bottom to the top of the ultracentrifuge tube using a long needle; then place it on the gradient mixer, select the 15%-45% sucrose gradient preparation program, and run continuously for 113 seconds to obtain a 15%-45% continuous gradient sucrose solution; S2, Sucrose density gradient ultracentrifugation: The PRRS virus particle sample was centrifuged at 8000 rpm for 3 min at 4℃, and the supernatant was collected for later use; 1.0 ml of sucrose continuous gradient solution was discarded, and then 1.5 ml of supernatant was slowly added to the top layer. The sample was centrifuged horizontally at 35000 rpm at 10℃ for 3.5 h to obtain the centrifuged sample. S3. Preparation of the high performance liquid chromatograph: Connect the pump head of the high performance liquid chromatograph to the external mobile phase (45% sucrose solution) with a tubing with a 15cm needle tip and a diameter of 0.5mm. Turn on the pumping system and ultraviolet detection system of the high performance liquid chromatograph and balance the baseline. S4. Detection of PRRS virus particle content: Insert the long needle from S3 into the bottom of the ultracentrifuge tube containing the centrifuged sample; set the flow rate to 1.0 ml / min and the detection wavelength to 280 nm. The computer system of the chromatograph will automatically draw the chromatogram, and manual integration will be performed to obtain the target peak area. S5. Prepare standard solutions of thyroglobulin standard protein in six concentration gradients within the concentration range of 10-160 µg / ml using TN buffer (pH 8.0); Take 1.5 ml of standard solution sample, follow S1-S3 procedures, and after baseline equilibration, insert the long needle into the bottom of the ultracentrifuge tube containing the standard solution sample; run at a flow rate of 1.0 ml / min, detect at 280 nm UV wavelength, integrate the absorption peak of the chromatogram to obtain the peak area, and establish a linear regression equation between the absorption peak area and protein concentration. S6. Based on the linear regression equation between protein concentration and peak area established in S5, substitute the target absorption peak area of ​​PRRS virus particles into the regression equation to calculate the content of PRRS virus particles in the sample to be tested.

2. The method for quantitatively detecting PRRS virus particles according to claim 1, characterized in that: In S1, before using the gradient mixer, it should be adjusted to balance using a level.

3. The method for quantitatively detecting PRRS virus particles according to claim 1, characterized in that: In step S3, deionized water is used as the mobile phase to remove air bubbles from the system.

4. The application of the method for quantitative detection of PRRS virus particles according to any one of claims 1 to 3 in evaluating the antigen content of PRRS vaccines.

5. The application of the method for quantitative detection of PRRS virus particles according to any one of claims 1 to 3 in the preparation of PRRS vaccines.

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