A method for quantitatively detecting SVA virus particles and its application
The detection of SVA virus particles through sucrose density gradient ultracentrifugation and high performance liquid chromatography system solves the problems of low accuracy and high cost of existing detection methods, and achieves efficient and accurate detection of viral particle content.
Patent Information
- Application Number
- CN202411927759.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-25
- Publication Date
- 2025-04-04
AI Technical Summary
The existing SVA virus particle content detection methods have low accuracy, poor repeatability, long cycle, high labor costs and operating costs, and it is impossible to accurately judge the final immune dose of the vaccine.
The sucrose density gradient ultracentrifugation method combined with high performance liquid chromatography system was used to prepare sucrose gradient solution and ultracentrifuge separation. The optical signal of the isolated SVA virus particles was detected by ultraviolet and lasers, and the content of the virus particles was calculated based on the ultraviolet absorption peak area of the thyroglobulin standard.
It improves the accuracy and repetition of the inspection, shortens the inspection cycle, reduces labor and operating costs, and overcomes the complex operation and high cost problems of traditional methods.
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Figure CN119757154A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of quantitative analysis, and in particular to a method and application for quantitatively detecting SVA virus particles. Background Art
[0002] Seneca virus disease can cause blisters and ulcerated wounds on the nose and hoof crown of pigs, occasionally fever and diarrhea, and the mortality rate of newborn piglets 1-3 days old can reach 30%-70%, causing significant economic losses to the pig industry. The disease is caused by Senecavirus (SVA). Swine Seneca virus disease is a newly emerging animal infectious disease in my country. Although it is currently in a sporadic state, there is no commercial vaccine that can provide an effective means of prevention, and there is no specific drug. As the virus continues to mutate and recombine, more and more new strains have been found to be pathogenic in recent years, and the pathogenicity is becoming stronger and stronger, and there is a possibility of a large-scale outbreak.
[0003] The accurate quantification of SVA virus particles during the vaccine development process can guide the determination and improvement of vaccine production processes, thereby improving production efficiency and saving costs; the content of virus particles in the vaccine is directly related to the final immune protection effect and safety of the vaccine. At present, the detection of SVA virus liquid content mainly adopts the cell half infection dose TCID50 and real-time fluorescence quantitative PCR, but the sensitivity, accuracy and repeatability of these two methods are difficult to guarantee. At present, the quantitative method of SVA inactivated virus liquid in industrial production is still blank, and it is impossible to accurately determine the final immune dose of the vaccine, which increases the difficulty of controlling the antigen content in the vaccine preparation process. Sucrose density gradient centrifugation is a classic method for separating viruses. Sucrose is relatively mild and is conducive to maintaining the integrity of virus particles. However, the traditional sucrose density gradient centrifugation method is all manually operated, the process is cumbersome, the repeatability is poor, the cycle is long, and the labor cost and operation cost are high, which greatly reduces the accuracy of sucrose density gradient centrifugation for quantitative SVA. Therefore, it is of great application value to establish a more accurate and simple method for detecting the content of SVA virus particles. Summary of the invention
[0004] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a method and application for quantitatively detecting SVA virus particles, so as to solve the problems of low accuracy, poor repeatability, long cycle, high labor cost and high operating cost of the existing detection method of SVA virus particle content.
[0005] The technical solution of the present invention to solve the above technical problem is as follows: a method for quantitatively detecting SVA virus particles is provided, comprising the following steps: (1) Preparation of sucrose gradient solution; (2) Separation of SVA virus using sucrose density gradient ultracentrifugation; (3) detecting the optical signal of the SVA virus separated in step (2) by ultraviolet and laser; (4) Calculate the content of SVA virus particles based on the regression equation of the ultraviolet absorption peak area of thyroglobulin standard and protein concentration.
[0006] Furthermore, the preparation of the sucrose gradient solution in step (1) includes the following steps: preparing 20 wt% and 40 wt% sucrose solutions respectively with TNE buffer, marking an ultracentrifuge tube, then first adding 20 wt% sucrose solution to the marked position of the ultracentrifuge tube, then adding 40 wt% sucrose solution from the bottom of the ultracentrifuge tube to the top, selecting a 20-40 wt% sucrose gradient preparation program, working for 100-150 s, and obtaining.
[0007] Furthermore, step (2) sucrose density gradient ultracentrifugation includes the following steps: centrifuging the SVA virus particle sample at 8000-10000 rpm for 3-10 min, taking the supernatant for later use; taking the supernatant and then adding the sucrose gradient solution described in step (1), and then centrifuging at 30000-40000 rpm for 3-4 h to obtain a centrifuged sample.
[0008] Furthermore, in step (3), deionized water and 45% sucrose solution were used as mobile phases, the flow rate was 1.0-2.0 mL / min, and the detection wavelength was set to 280 nm.
[0009] Furthermore, the regression equation is: C=(0.00004X+2.3781) / 1.5; where C is the protein concentration and X is the absorption peak area.
[0010] The present invention also provides an application of the method for quantitatively detecting SVA virus particles in the preparation of SVA vaccines.
[0011] The present invention has the following beneficial effects: The detection method of the present invention comprises the following steps: (1) preparation of sucrose gradient solution; (2) separation of SVA virus by sucrose density gradient ultracentrifugation; (3) detection of the optical signal of SVA virus separated in step (2) by ultraviolet and laser; (4) calculation of the content of SVA virus particles according to the regression equation of the ultraviolet absorption peak area of thyroglobulin standard and protein concentration. In the present invention, the sucrose density gradient is prepared by a fully automatic gradient instrument, and the peak of SVA virus particles is fully automatically analyzed by a high performance liquid chromatography system. A standard curve is established by a known concentration of standard protein, and the sample to be tested is calculated, which overcomes the problems of poor repeatability and complex operation process of the traditional sucrose density gradient ultracentrifugation method, and has the advantages of short detection cycle, high sensitivity, good repeatability, small batch error, etc. The present invention has important guiding significance for the development, production process and quality control of SVA vaccine in my country. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 This is the sucrose density gradient ultracentrifugation chromatographic analysis diagram of thyroglobulin standard protein; Figure 2 The figure is the linear relationship between the absorption peak area and concentration of thyroglobulin at different dilution concentrations; Figure 3 This is the chromatographic analysis spectrum of the SVA virus particle sample detected by sucrose density gradient ultracentrifugation combined with high performance liquid chromatography in Example 2; Figure 4 This is a transmission electron microscope analysis result diagram of the target collection peak of the high performance liquid chromatograph in Example 2; Figure 5 This is a graph showing the PCR detection results of the target collection peak of the high performance liquid chromatograph in Example 2; Figure 6 This is a graph showing the results of chromatographic analysis of the SVA virus particle sample of Example 2-4 after sucrose density gradient ultracentrifugation. DETAILED DESCRIPTION
[0013] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.
[0014] Example 1 Establishment of standard curve Prepare 6 standard solution samples with a concentration gradient of 10-160 µg / mL using TNE buffer (pH 8.0) for thyroglobulin standard protein and store at 2-8°C for later use; 1. Preparation of sucrose gradient: (1) Prepare 20 wt% and 40 wt% sucrose solutions using TNE buffer (pH 8.0); (2) Take a 12.5 mL ultracentrifuge tube and mark it using the scale provided on the gradient mixer. (3) First, add 20% sucrose solution to the ultracentrifuge tube to the mark, and then use a long needle to add 40% sucrose solution from the bottom to the top of the ultracentrifuge tube. Do not generate bubbles during the whole process. (4) Turn on the gradient mixer and adjust the gradient mixer to balance using a level. Place the ultracentrifuge tube containing the sucrose gradient solution in the gradient mixer, select the 20 wt%-40 wt% sucrose gradient preparation program, and work continuously for 115 s to prepare a 20 wt%-40 wt% continuous gradient sucrose solution.
[0015] 2. Sucrose density gradient ultracentrifugation: (1) Take the thyroglobulin standard protein, centrifuge it at 8000 rpm for 3 min at 4°C, and take the supernatant for later use; (2) Aspirate and discard 1.0 mL of the prepared sucrose gradient solution, add 1.5 mL of the supernatant, and centrifuge horizontally at 10°C and 35,000 rpm for 3.5 h to obtain the centrifuged sample; 3. Preparation of HPLC: (1) Connect the pump head of the HPLC to the external mobile phase using a 0.5 mm diameter tube with a 15 cm long needle at the end; (2) Turn on the pumping system and UV detection system of the HPLC and use deionized water as the mobile phase to remove the bubbles in the system; (3) Use 45% sucrose solution as the mobile phase to balance the baseline.
[0016] 4. Determination of thyroglobulin standard protein: (1) Insert the long needle in step 3 into the bottom of the ultracentrifuge tube in step 2; (2) Turn on the pump system, set the flow rate to 1.0 mL / min, set the detection wavelength to 280 nm, and use the chromatograph's computer system to automatically draw the chromatogram. Perform manual integration to obtain the target peak area.
[0017] like Figure 1As shown in the figure, the thyroglobulin standard protein shows a chromatographic absorption peak. The concentrations of the diluted standard solution samples in the experiment are 0µg / mL, 10µg / mL, 20µg / mL, 40µg / mL, 80µg / mL and 160µg / mL respectively. The absorption peaks of the standard proteins of different concentrations are integrated to obtain the peak area. The linear response trend line is obtained with the peak area as the abscissa and the protein content as the ordinate. It can be concluded that C=(0.00004X+2.3781) / 1.5; where C is the protein concentration in µg / mL, and X is the absorption peak area in mV.min, see Figure 2 The R of the obtained standard curve 2 The value is 0.9989, which shows that this method has high accuracy.
[0018] Example 2 A method for quantitatively detecting SVA virus particles comprises the following steps: 1. Preparation of sucrose gradient: (1) Prepare 20 wt% and 40 wt% sucrose solutions using TNE buffer (pH 8.0); (2) Take a 12.5 mL ultracentrifuge tube and mark it using the scale provided on the gradient mixer. (3) First, add 20% sucrose solution to the ultracentrifuge tube to the mark, and then use a long needle to add 40% sucrose solution from the bottom to the top of the ultracentrifuge tube. Do not generate bubbles during the whole process. (4) Turn on the gradient mixer and adjust the gradient mixer to balance using a level. Place the ultracentrifuge tube containing the sucrose gradient solution in the gradient mixer, select the 20 wt%-40 wt% sucrose gradient preparation program, and work continuously for 115 s to prepare a 20 wt%-40 wt% continuous gradient sucrose solution.
[0019] 2. Sucrose density gradient ultracentrifugation: (1) Take the SVA virus particle sample (SVA-1), centrifuge it at 8000 rpm for 3 min at 4°C, and take the supernatant for later use; (2) Aspirate and discard 1.0 mL of the prepared sucrose gradient solution, add 1.5 mL of the supernatant, and centrifuge horizontally at 10°C and 35,000 rpm for 3.5 h to obtain a centrifuged sample; 3. Preparation of HPLC: (1) Connect the pump head of the HPLC to the external mobile phase using a 0.5 mm diameter tube with a 15 cm long needle at the end; (2) Turn on the pumping system and UV detection system of the HPLC and use deionized water as the mobile phase to remove the bubbles in the system; (3) Use 45% sucrose solution as the mobile phase to balance the baseline.
[0020] 4. Determination of SVA virus particle content: (1) Insert the long needle in step 3 into the bottom of the ultracentrifuge tube in step 2; (2) Turn on the pump system, set the flow rate to 1.0 mL / min, set the detection wavelength to 280 nm, and use the chromatograph's computer system to automatically draw the chromatogram. Perform manual integration to obtain the target peak area. (3) When the chromatograph is detecting, open the fraction collector, collect the target peak position, and store it in the collection tube at 4°C for later use.
[0021] The chromatogram drawn from the measured data is as follows Figure 3 As shown in A, the chromatographic peak of the SVA virus particle sample appeared at 4-6 min; no chromatographic peak appeared after centrifugation of the cell culture sample that was not inoculated with SVA virus (see Figure 3 B).
[0022] The target peak sample collected in step 4 was observed under transmission electron microscopy, and complete SVA virus particles were observed, such as Figure 4 The target peak samples collected in step 4 were subjected to PCR detection, and clear specific amplification bands were observed, as shown in Figure 5 shown.
[0023] Example 3 A method for quantitatively detecting SVA virus particles comprises the following steps: 1. Preparation of sucrose gradient: (1) Prepare 20 wt% and 40 wt% sucrose solutions using TNE buffer (pH 8.0); (2) Take a 12.5 mL ultracentrifuge tube and mark it using the scale provided on the gradient mixer. (3) First, add 20% sucrose solution to the ultracentrifuge tube to the mark, and then use a long needle to add 40wt% sucrose solution from the bottom to the top of the ultracentrifuge tube. Do not generate bubbles during the whole process. (4) Turn on the gradient mixer and adjust the gradient mixer to balance using a level. Place the ultracentrifuge tube containing the sucrose gradient solution in the gradient mixer, select the 20 wt%-40 wt% sucrose gradient preparation program, and work continuously for 115 s to prepare a 20 wt%-40 wt% continuous gradient sucrose solution.
[0024] 2. Sucrose density gradient ultracentrifugation: (1) Take the SVA virus particle sample (SVA-2), centrifuge it at 10,000 rpm for 5 min at 4°C, and take the supernatant for later use; (2) Discard 2.0 mL of the prepared sucrose gradient solution, add 1.5 mL of the supernatant, and centrifuge horizontally at 10°C and 35,000 rpm for 3.5 h to obtain a centrifuged sample; 3. Preparation of HPLC: (1) Connect the pump head of the HPLC to the external mobile phase using a 0.5 mm diameter tube with a 15 cm long needle at the end; (2) Turn on the pumping system and UV detection system of the HPLC and use deionized water as the mobile phase to remove the bubbles in the system; (3) Use 45% sucrose solution as the mobile phase to balance the baseline.
[0025] 4. Determination of SVA virus particle content: (1) Insert the long needle in step 3 into the bottom of the ultracentrifuge tube in step 2; (2) Turn on the pump system, set the flow rate to 1.0 mL / min, set the detection wavelength to 280 nm, and use the chromatograph's computer system to automatically draw the chromatogram. Perform manual integration to obtain the target peak area.
[0026] Example 4 A method for quantitatively detecting SVA virus particles comprises the following steps: 1. Preparation of sucrose gradient: (1) Prepare 20 wt% and 40 wt% sucrose solutions using TNE buffer (pH 8.0); (2) Take a 12.5 mL ultracentrifuge tube and mark it using the scale provided on the gradient mixer. (3) First, add 20% sucrose solution to the ultracentrifuge tube to the mark, and then use a long needle to add 40wt% sucrose solution from the bottom to the top of the ultracentrifuge tube. Do not generate bubbles during the whole process. (4) Turn on the gradient mixer and adjust the gradient mixer to balance using a level. Place the ultracentrifuge tube containing the sucrose gradient solution in the gradient mixer, select the 20 wt%-40 wt% sucrose gradient preparation program, and work continuously for 115 s to prepare a 20 wt%-40 wt% continuous gradient sucrose solution.
[0027] 2. Sucrose density gradient ultracentrifugation: (1) Take the SVA virus particle sample (SVA-3), centrifuge it at 10,000 rpm for 10 min at 4°C, and take the supernatant for later use; (2) Aspirate and discard 1.0 mL of the prepared sucrose gradient solution, add 1.5 mL of the supernatant, and centrifuge horizontally at 10°C and 35,000 rpm for 3.5 h to obtain a centrifuged sample; 3. Preparation of HPLC: (1) Connect the pump head of the HPLC to the external mobile phase using a 0.5 mm diameter tube with a 15 cm long needle at the end; (2) Turn on the pumping system and UV detection system of the HPLC and use deionized water as the mobile phase to remove the bubbles in the system; (3) Use 45% sucrose solution as the mobile phase to balance the baseline.
[0028] 4. Determination of SVA virus particle content: (1) Insert the long needle in step 3 into the bottom of the ultracentrifuge tube in step 2; (2) Turn on the pump system, set the flow rate to 1.5 mL / min, set the detection wavelength to 280 nm, and use the chromatograph's computer system to automatically draw the chromatogram. Perform manual integration to obtain the target peak area.
[0029] The SVA virus particle sample of Example 2-4 was tested according to the determination method and then the target peak was integrated. Then the target absorption peak area was substituted into the standard curve C = (0.00004X + 2.3781) / 1.5 to calculate the antigen concentration, where C is the sample protein concentration in µg / mL, X is the absorption peak area in mV.min, and the results are shown in Figure 6 The measurement results are shown in Table 1.
[0030] Table 1 Determination of the content of SVA virus particle samples in Examples 2-4
[0031] The above results show that the quantitative detection method of the present invention is used to detect the SVA virus particle content. The peak position of the SVA virus particles is consistent and has repeatability.
[0032] 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, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for quantitatively detecting SVA virus particles, characterized in that: The following steps are involved: (1) Preparation of sucrose gradient solution; (2) Separation of SVA virus using sucrose density gradient ultracentrifugation; (3) detecting the optical signal of the SVA virus separated in step (2) by ultraviolet and laser; (4) Calculate the content of SVA virus particles based on the regression equation of the ultraviolet absorption peak area of thyroglobulin standard and protein concentration.
2. The method for quantitatively detecting SVA virus particles according to claim 1, characterized in that: The preparation of the sucrose gradient solution in step (1) comprises the following steps: preparing 20wt% and 40wt% sucrose solutions respectively with TNE buffer, marking an ultracentrifuge tube, then first adding 20wt% sucrose solution to the marked position of the ultracentrifuge tube, then adding 40wt% sucrose solution from the bottom of the ultracentrifuge tube to the top, selecting a 20-40wt% sucrose gradient preparation program, working for 100-150s, and obtaining the obtained solution.
3. The method for quantitatively detecting SVA virus particles according to claim 1, characterized in that: The sucrose density gradient ultracentrifugation in step (2) comprises the following steps: centrifuging the SVA virus particle sample at 8000-10000 rpm for 3-10 min, taking the supernatant, and then adding the supernatant to the sucrose gradient solution in step (1), and centrifuging at 30000-40000 rpm for 3-4 h to obtain a centrifuged sample.
4. The method for quantitatively detecting SVA virus particles according to claim 1, characterized in that: In the step (3), deionized water and 45% sucrose solution are used as the mobile phase, the flow rate is 1.0-2.0 mL / min, and the detection wavelength is set to 280 nm.
5. The method for quantitatively detecting SVA virus particles according to claim 1, characterized in that: The regression equation is: C=(0.00004X+2.3781) / 1.5; wherein C is the protein concentration and X is the absorption peak area.
6. Use of the method for quantitatively detecting SVA virus particles according to any one of claims 1 to 5 in the preparation of SVA vaccines.