A method for quantitatively detecting feline triple virus content and its application

Abstract

The present invention discloses a method and application for quantitatively detecting the content of feline triple virus, and belongs to the field of quantitative analysis technology. The detection method of the present invention comprises the following steps: (1) preparing a sucrose gradient solution; (2) taking the supernatant after centrifuging the virus particle sample; aspirating the top solution of the sucrose gradient solution prepared in step (1), then adding the supernatant, and preparing the centrifuged sample after centrifugation; (3) determining the content of the centrifuged sample by high performance liquid chromatography. The present invention utilizes a high performance liquid chromatography system to realize the fully automatic analysis of the peaks of FPV, FHV, and FCV virus particles, establishes a standard curve through standard proteins, and calculates the content of the sample to be tested. The method has the advantages of easy popularization and mastery of operation, good repeatability, high accuracy, and small batch-to-batch differences, and can meet the use needs of personnel at different levels. The present invention has broad application prospects in the production process and quality assessment of cat triple vaccines in my country.

Description

Technical Field

[0001] The invention relates to the technical field of quantitative analysis, and in particular to a method for quantitatively detecting the content of feline triple virus and an application thereof. Background Art

[0002] Feline panleukopenia, also known as feline distemper or feline infectious enteritis, is an acute contact infectious disease characterized by biphasic fever, significant leukopenia, vomiting, diarrhea, and severe dehydration, with high morbidity and mortality. The pathogen of the disease is feline panleukopenia virus (FPV), which belongs to the Parvoviridae family and the Parvovirus genus. It is a non-enveloped single-stranded positive-strand DNA virus with an icosahedral symmetric capsid, spherical, and a diameter of 20~24nm.

[0003] Feline infectious rhinobronchitis is one of the most serious respiratory infectious diseases in cats caused by feline herpesvirus type 1 (FHV), with fever, sneezing, coughing, keratoconjunctivitis, nasal and ocular serous or purulent discharge, and periocular skin ulcers as typical symptoms. FHV belongs to the family Herpesviridae, subfamily Alphaherpesvirinae, and is a linear single-stranded DNA virus with an envelope. It is a large virus with a protomorph or elliptical shape and icosahedral symmetry. The diameter is about 128~168nm.

[0004] The pathogen of feline calicivirus disease is feline calicivirus (FCV), which belongs to the genus Herpesvirus in the family Caliciviridae. It is a non-enveloped single-stranded positive-sense RNA virus with a diameter of approximately 35~39nm. It often co-infects with other viruses and causes oral lesions, upper respiratory tract infections, gastrointestinal infections and malignant systemic diseases in cats.

[0005] The above three diseases seriously threaten the health of pets due to their high infection rates and low cure rates. The most effective and economical measure to prevent and control their spread is vaccination. Among the many types of vaccines, attenuated vaccines have better immune effects, but there is a potential risk of reversion to virulence, so FPV, FHV, and FCV triple inactivated vaccines still dominate the Chinese market. In vaccine production, the content of antigens is directly related to the final immune protection effect and safety of the vaccine, so accurate quantification of antigens is the top priority in the development and production of cat triple vaccines.

[0006] Common detection methods for FPV, FHV, and FCV viruses include hemagglutination test and hemagglutination inhibition test, enzyme-linked immunosorbent assay, latex agglutination test, virus plaque test, immunoenzyme staining method, and immunofluorescence method, but these methods have complex operating conditions, poor repeatability, and certain limitations in sensitivity and accuracy. Therefore, a more accurate and simple method for detecting the content of FPV, FHV, and FCV virus particles is urgently needed to accurately evaluate and control the quality of vaccines, thereby guiding the development and industrial production of cat triple vaccines. Summary of the invention

[0007] 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 the content of feline triple virus, so as to solve the problems of poor repeatability, sensitivity and accuracy of the existing method for detecting the content of feline triple virus.

[0008] The technical solution of the present invention to solve the above technical problems is as follows: a method for quantitatively detecting the content of feline triple virus is provided, comprising the following steps: (1) Prepare sucrose gradient solution; (2) Centrifuge the virus particle sample and take the supernatant; take the top solution of the sucrose gradient solution prepared in step (1), then add the supernatant, and centrifuge to obtain a centrifuged sample; (3) Determine the content of the centrifuged sample by high performance liquid chromatography.

[0009] Furthermore, step (1) preparing the sucrose gradient solution is specifically as follows: 15 wt% and 45 wt% sucrose solutions are prepared respectively with TNE buffer, an ultracentrifuge tube is marked, and then 15 wt% sucrose solution is first added to the marked portion of the ultracentrifuge tube, and then 45 wt% sucrose solution is added from the bottom of the ultracentrifuge tube to the top, a 15-45 wt% sucrose gradient preparation program is selected, and the mixture is worked for 100-150 s to obtain the obtained solution.

[0010] Furthermore, in step (2), the centrifugation rate of the virus particle sample is 8000-10000 rpm, and the centrifugation time is 3-5 min; the centrifugation rate after adding the supernatant is 30000-40000 rpm, and the centrifugation time is 3.5-4 h.

[0011] Furthermore, the mobile phase of step (3) is deionized water and 45% sucrose solution, the flow rate is 1.0-2.0 mL / min, and the detection wavelength is 280 nm.

[0012] Furthermore, in step (3), the centrifugal sample content is determined by high performance liquid chromatography, and then the centrifugal sample concentration is calculated by a linear regression equation between the standard protein concentration and the absorption peak area.

[0013] Furthermore, the linear regression equation is: C=(0.00004X+4.0176) / 1.5; where C is the sample protein concentration and X is the absorption peak area.

[0014] The present invention also provides an application of the method for quantitatively detecting the content of feline triple virus in the preparation of a feline triple vaccine.

[0015] The present invention has the following beneficial effects: The detection method of the present invention comprises the following steps: (1) preparing a sucrose gradient solution; (2) taking the supernatant after centrifuging the virus particle sample; taking the top solution of the sucrose gradient solution prepared in step (1), then adding the supernatant, and preparing a centrifuged sample after centrifugation; (3) determining the content of the centrifuged sample by high performance liquid chromatography. In the present invention, the sucrose density gradient is prepared by a fully automatic gradient instrument, and the high performance liquid chromatography system is used to realize the fully automatic analysis of the peaks of FPV, FHV, and FCV virus particles. The standard curve is established by the standard protein, and the content of the sample to be tested is calculated. The method has the advantages of easy operation, good repeatability, high accuracy, and small batch-to-batch difference, and can meet the use needs of people at different levels. The present invention has broad application prospects in the production process and quality assessment of cat triple vaccines in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a chromatographic analysis diagram of thyroglobulin standard protein after sucrose density gradient ultracentrifugation; Figure 2 The figure is the linear relationship between the absorption peak area and concentration of thyroglobulin at different dilution concentrations; Figure 3 Chromatographic analysis of FPV, FHV, and FCV virus particle samples detected by sucrose density gradient ultracentrifugation combined with high performance liquid chromatography; Figure 4 Transmission electron microscopy analysis results of the target collection peaks of the HPLC; Figure 5 This is the chromatographic analysis profile of the FPV virus particle sample after sucrose density gradient ultracentrifugation; Figure 6 This is the chromatographic analysis profile of the FHV virus particle sample after sucrose density gradient ultracentrifugation; Figure 7 This is a chromatographic analysis graph of an FCV virus particle sample after sucrose density gradient ultracentrifugation. 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.

[0017] Example 1 Establishment of standard curve Prepare 6 standard solution samples with a concentration gradient of 5-80 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 15 wt% and 45 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 15% sucrose solution to the ultracentrifuge tube to the mark, and then use a long needle to add 45% 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 15 wt%-45 wt% sucrose gradient preparation program, and work continuously for 115 s to prepare a 15 wt%-45 wt% continuous gradient sucrose solution.

[0018] 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) Pipette 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 HPLC pump head 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.

[0019] 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.

[0020] Depend on Figure 1It can be seen that the thyroglobulin standard protein will have an obvious chromatographic absorption peak. The concentrations of the diluted standard solution samples in the experiment are 0µg / mL, 5µg / mL, 10µg / mL, 20µg / mL, 40µg / mL and 80µg / mL. 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 horizontal coordinate and the protein content as the vertical coordinate. It can be obtained that C=(0.00004X+4.0176) / 1.5; where: C is the protein concentration, in µg / mL; X is the absorption peak area, in mV·min; the R2 value of the obtained standard curve is 0.9997, indicating that this curve has a high accuracy. The standard curve is shown in Figure 2 .

[0021] Embodiment 2: A method for quantitatively detecting the content of feline triple virus comprises the following steps: 1. Preparation of sucrose gradient: (1) Prepare 15 wt% and 45 wt% sucrose solutions using TNE buffer (pH 8.0); (2) Take a 12.5 mL ultracentrifuge tube and mark the scale of the ultracentrifuge tube using the scale provided by the gradient mixer; (3) First, add 15wt% sucrose solution to the ultracentrifuge tube to the scale, then use a long needle to slowly insert it into the bottom of the centrifuge tube, and gradually inject 45wt% sucrose solution to the top of the centrifuge tube, and cover the tube cap. Do not generate bubbles during the whole process; (4) Turn on the gradient mixer, adjust the gradient mixer to a horizontal level with a level, place the ultracentrifuge tube containing the sucrose gradient solution on the gradient mixer, select the 15 wt%-45 wt% sucrose gradient preparation program, and work continuously for 113 s to obtain a 15 wt%-45 wt% continuous gradient sucrose solution; 2. Sucrose density gradient ultracentrifugation: (1) Centrifuge the FPV, FHV, and FCV virus particle samples at 4°C, 8000 rpm for 3 min, and obtain the supernatant for later use; (2) First, discard 1.0 mL of the prepared sucrose gradient top solution, then slowly add 1.5 mL of each of the above supernatants to the top layer, centrifuge the sucrose solution containing FPV virus particles at 10°C and 35,000 rpm for 3.5 h, centrifuge the sucrose solution containing FHV virus particles at 10°C and 40,000 rpm for 3.5 h, and centrifuge the sucrose solution containing FCV virus particles at 10°C and 30,000 rpm for 3.5 h to obtain centrifuged samples; 3. Preparation of HPLC: (1) Use a 0.5 mm diameter tubing with a 15 cm long needle at the end to connect the external mobile phase (45 wt % sucrose solution) to the pump head of the HPLC; (2) Start the pumping system and UV detection system of the HPLC; (3) Use deionized water as the mobile phase to exhaust the system; (4) Use 45 wt % sucrose solution as the mobile phase, set the flow rate to 1.0 mL / min, and balance the baseline.

[0022] 4. FPV, FHV, FCV virus particle sample detection: (1) Slowly insert the long needle in step 3 into the bottom of the ultracentrifuge sample tube in step 2; (2) Start the pump system, set the detection wavelength to 280 nm, and start automatic detection at a flow rate of 1.0 mL / min. The computer system of the chromatograph automatically draws the sample chromatogram in real time; (3) Manually integrate the chromatogram to obtain the target peak area; The chromatogram drawn is as follows Figure 3 As shown, the chromatographic peak position of the FPV virus particle sample is 5.5-7.5min ( Figure 3 A). The peak position of the FHV virus particle sample chromatographic peak is 2-3.5min ( Figure 3 B). The peak position of the FCV virus particle sample chromatographic peak is 5-6.5min ( Figure 3 C).

[0023] 5. Open the fraction collector, collect samples from each target peak position, and store them at 2-8°C for later use.

[0024] 6. Use transmission electron microscopy to observe the target peak samples collected in step 5, and you can see complete FPV, FHV, and FCV virus particles, such as Figure 4 shown.

[0025] 7. Performing fluorescence quantitative PCR detection on each target peak sample collected in step 5 can detect FPV, FHV, and FCV. The fluorescence quantitative PCR detection results of each target peak sample are shown in Table 1; Table 1 Fluorescence quantitative PCR detection results Sample name Cq value Positive Control 11.64 Negative control N / A FPV target peak collection sample 14.44 FHV target peak collection sample 18.92 FCV target peak collection sample 21.84 Integrate the absorption peaks of FPV, FHV, and FCV virus particle samples: After testing three virus particle samples of FPV, FHV, and FCV according to the method of the present invention, manually integrate the target peaks. Calculate the content of FPV, FHV, and FCV virus particle samples: Substitute the target absorption peak area into the calculation formula C=(0.00004X+4.0176) / 1.5 obtained in Example 1 to calculate the measured antigen concentration, where C is the protein concentration of the measured sample, in µg / mL, and X is the absorption peak area, in mV·min.

[0026] The statistical results of the detection of FPV, FHV, and FCV virus particle antigen content are shown in Table 2; the chromatograms of 3 batches of different FPV antigens detected by sucrose density gradient centrifugation combined with high performance liquid chromatography are shown in Figure 5 ; Chromatograms of 3 batches of different FHV antigens detected by sucrose density gradient centrifugation combined with high performance liquid chromatography are shown in Figure 6 ; Chromatograms of 3 batches of different FCV antigens detected by sucrose density gradient centrifugation combined with high performance liquid chromatography are shown in Figure 7 .

[0027] Table 2 Results of detection of antigen content of FPV, FHV and FCV virus particles

[0028] Table 2 Statistical results of antigen content and Figure 5-7 It can be seen from the chromatogram that the peak positions of FPV, FHV and FCV virus particles in different batches are slightly different, which proves that the use of sucrose density gradient centrifugation combined with high performance liquid chromatography quantitative method to detect the content of FPV, FHV and FCV virus particles has good feasibility.

[0029] 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 the content of feline triple virus, characterized in that, The following steps are involved: (1) Prepare sucrose gradient solution; (2) Centrifuging the virus particle sample and taking the supernatant; aspirating the top solution of the sucrose gradient solution prepared in step (1), then adding the supernatant, and centrifuging to obtain a centrifuged sample; (3) Determine the content of the centrifuged sample by high performance liquid chromatography.

2. The method for quantitatively detecting feline triple virus content according to claim 1, characterized in that, The step (1) of preparing the sucrose gradient solution specifically comprises: preparing 15wt% and 45wt% sucrose solutions respectively with TNE buffer, marking the ultracentrifuge tube, then first adding the 15wt% sucrose solution to the marked position of the ultracentrifuge tube, and then adding the 45wt% sucrose solution from the bottom of the ultracentrifuge tube to the top, selecting the 15-45wt% sucrose gradient preparation program, working for 100-150s, and obtaining the result.

3. The method for quantitatively detecting feline triple virus content according to claim 1, characterized in that, The centrifugation rate of the virus particle sample in step (2) is 8000-10000 rpm, and the centrifugation time is 3-5 min; the centrifugation rate after adding the supernatant is 30000-40000 rpm, and the centrifugation time is 3.5-4 h.

4. The method for quantitatively detecting feline triple virus content according to claim 1, characterized in that, The mobile phase of step (3) is deionized water and 45% sucrose solution, the flow rate is 1.0-2.0 mL / min, and the detection wavelength is 280 nm.

5. The method for quantitatively detecting the content of feline triple virus according to claim 1, characterized in that, In the step (3), the centrifugal sample content is determined by high performance liquid chromatography, and then the centrifugal sample concentration is calculated by a linear regression equation between the standard protein concentration and the absorption peak area.

6. The method for quantitatively detecting the content of feline triple virus according to claim 5, characterized in that, The linear regression equation is: C=(0.00004X+4.0176) / 1.5; wherein C is the sample protein concentration, and X is the absorption peak area.

7. Application of the method for quantitatively detecting the content of feline triple virus according to any one of claims 1 to 6 in the preparation of a feline triple vaccine.

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