Preparation, identification and quality evaluation method of standard sample of antibody-negative serum for common diseases of pigs

Abstract

This invention discloses a method for preparing, identifying, and evaluating the quality of SPF (Porcine Common Disease Antibody-Negative Serum Standard Samples). This invention belongs to the field of biological detection. The method for preparing SPF porcine negative serum standard samples includes the following steps: (1) raw material preparation and initial screening; (2) serum testing; (3) negative serum confirmation; (4) standard sample preparation; (5) homogeneity testing and evaluation; and (6) stability testing and evaluation, ultimately obtaining SPF porcine common disease antibody-negative serum standard samples. The standard samples prepared by the above method can be directly used as quality control materials and negative controls for serological detection of various common porcine diseases. They can be used for the research and development and quality control of detection kits, as well as for kit comparison and proficiency testing. This fills the gap in standardized negative serum samples in this field in my country, helps to standardize the detection process, and improves the comparability of detection results.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to a method for preparing, identifying, and evaluating the quality of standard serum samples with negative antibodies against common swine diseases. Background Technology

[0002] African swine fever (ASF) is an acute, highly contagious, and deadly disease caused by the African swine fever virus (ASFV). Currently, global control of this disease relies primarily on a comprehensive approach including diagnosis, surveillance, culling, and enhanced biosecurity management. Early and accurate diagnosis of ASF is crucial for identifying the pathogen and implementing timely and effective measures. Serological testing is one of the key technologies for ASF surveillance, diagnosis, and control. Positive ASF virus sera or quality control samples are relatively easy to obtain, while negative sera standards serve as the core control material in testing, and their quality directly determines the accuracy, reliability, and comparability of the test results. However, there are currently no reports of the development of negative sera standards / materials or quality control samples for antibodies against ASF and other common swine diseases in my country, and there is no commercial production or supply of such products. This severely hinders the development of related testing work, reagent / kit comparison and evaluation, and the effective implementation and application of written standards such as national standards for ASF testing.

[0003] Currently, there is a severe shortage, or even a near-absence, of negative serum standard samples / standard substances / quality control products for serological testing of common swine diseases such as African swine fever in the Chinese market. Their development also faces numerous problems: First, the raw materials lack specific screening indicators, making it impossible to effectively eliminate potential contamination or cross-reaction risks; second, there is a lack of targeted quality control and monitoring markers, making it difficult to accurately control sample quality; third, the homogeneity and stability testing and evaluation system is incomplete, almost entirely absent in this area. Currently, only a single indicator is used for detection and evaluation, or even no testing and evaluation are conducted at all, leading to significant quality fluctuations in negative serum samples during storage, transportation, and use, affecting the consistency of test results. As mentioned above, the lack of physical negative serum standard samples and the deficiencies in the quality evaluation system have become the core bottleneck restricting the accuracy, scientific validity, and comparability of serological testing results for common swine diseases such as African swine fever. Therefore, there is an urgent need to develop high-quality negative swine serum standards and establish a standardized preparation and quality evaluation system to meet practical needs.

[0004] Unlike serum antibodies produced by pathogen infection or vaccine immunization, negative serum does not contain any antibodies induced by pathogens and therefore cannot be detected using any specific antigens. However, negative serum is still a protein, and its total protein content can be detected using the BCA method. Furthermore, healthy pig negative serum contains CD79A and CD79B proteins encoded by B cells, which form a heterodimer and are the core signal transduction subunit of the B cell receptor (BCR). In serum samples from healthy pigs, CD79 expression is "positive," making it an important specific marker tracer protein. Therefore, the content of negative serum can be indirectly evaluated by measuring total protein content using BCA and detecting the CD79 gene. Developing a standardized, high-quality method for preparing and assessing the quality of negative serum standard samples for common diseases such as African swine fever is of great practical significance for ensuring the scientific validity and authority of serological testing results for African swine fever and other swine diseases. Summary of the Invention

[0005] The core problem to be solved by this invention is how to develop antibody-negative serum standard samples applicable to various common swine diseases such as African swine fever, as well as their identification and quality assessment methods.

[0006] To address the aforementioned issues, this invention provides a method for preparing, identifying, and evaluating the quality of SPF porcine antibody-negative serum standard samples.

[0007] The method for preparing SPF porcine antibody-negative serum standard samples provided by this invention includes the following steps: (1) Raw material preparation and initial screening: Select healthy 40-day-old SPF boars, collect blood aseptically and separate serum, and after heat inactivation treatment and inactivation test, use multiple ELISA detection kits to detect serum antibodies against 10 kinds of swine diseases. The swine serum with negative test results is qualified SPF swine serum. (2) Serum testing: SPF pig serum that has passed the initial screening is subjected to sterility testing and mycoplasma testing to ensure that the serum is pure and uncontaminated; (3) Confirmation of negative serum: The mixed serum was repeatedly tested using ELISA and IIFA methods to confirm that all 10 swine disease antibodies were negative and had no cross-reactivity, thus obtaining negative serum raw materials; (4) Preparation of standard samples: The negative serum raw material is mixed with a freeze-drying protectant containing calf serum and cyclodextrin in an appropriate ratio and then freeze-dried. (5) Homogeneity test and evaluation: The total protein concentration was detected by BCA protein quantification kit and the CD79A gene mRNA level was detected by real-time PCR. The coefficient of variation (CV) was calculated. When the coefficient of variation CV < 5%, the sample homogeneity was deemed to be qualified. (6) Stability testing and evaluation: including repeated freeze-thaw stability, short-term stability and long-term stability testing and evaluation. When CV < 5%, the stability is deemed qualified and the negative serum standard sample of the common swine disease antibody is obtained.

[0008] Furthermore, the 10 common swine disease antibodies mentioned in step 1) are African swine fever virus antibody, foot-and-mouth disease virus type O antibody, classical swine fever virus antibody, porcine pseudorabies virus gB antibody, porcine reproductive and respiratory syndrome virus antibody, porcine circovirus type 2 antibody, porcine transmissible gastroenteritis virus antibody, porcine epidemic diarrhea virus antibody, porcine rotavirus antibody, and porcine parvovirus antibody.

[0009] In this paper, the serum collection and mixing methods are as follows: qualified SPF pigs are euthanized, all blood is collected and serum is separated, and multiple serum samples are mixed to ensure the representativeness of the raw materials; sterility tests are performed by culturing in TG and TSB media and mycoplasma detection to eliminate the risk of microbial contamination.

[0010] In this study, negative serum was confirmed by repeating the IIFA method on the mixed serum to further confirm that all 10 common swine disease antibodies were negative and there was no cross-reactivity, ensuring that the raw materials met the requirements of the negative serum standard sample.

[0011] Further, the aseptic treatment step described in step 2) is as follows: after mixing the separated serum, it is cultured in TG medium at 25°C and 37°C for 7 days, and simultaneously cultured in TSB medium at 25°C for 7 days, observing for colony growth daily; then mycoplasma testing is performed to confirm that there is no microbial contamination.

[0012] Furthermore, the GenBank accession number for the nucleotide sequence of the CD79A gene mentioned in step 5) is NM_001135962, and the update date is April 2, 2025.

[0013] Furthermore, the primers used in step 2) for the real-time PCR are upstream primer F and downstream primer R. The sequence of upstream primer F is 5'-CATGGATCTCTCAGAACTG-3', and the sequence of downstream primer R is 5'-GTTGACTTCGCACCTGTAC-3'. The PCR reaction also includes a probe with the sequence 5'-FAM-CATCCAGCAAGTGAACAAGAGCCA-BHQ1-3'.

[0014] In one specific embodiment, the reaction system for real-time PCR in step 2) comprises 0.5 μL of Go Script™ RT Mix, 10 μL of Go Taq Probe qPCR Master Mix (2×), 0.6 μL of upstream primer (10 pmol / μL), 0.6 μL of downstream primer (10 pmol / μL), 0.6 μL of probe (10 pmol / μL), 2.7 μL of nuclease-free water, and 5 μL of template; The PCR reaction conditions were: reverse transcription at 45°C for 30 min, pre-denaturation at 95°C for 5 min, followed by denaturation at 95°C for 5 s and annealing extension at 60°C for 30 s (with fluorescence collection), for a total of 35 cycles.

[0015] In the above method, step 3) is followed by a stability monitoring and evaluation step for the negative serum standard sample, which includes repeated freeze-thaw stability, short-term stability evaluation and long-term stability evaluation.

[0016] In one specific embodiment, the sampling quantity for repeated freeze-thaw stability is 3 standard samples, which are tested after 0, 3, and 5 freeze-thaw cycles, and each sample is tested 3 times. For short-term stability, the samples are stored at -20℃, 4℃, and 25℃ for 7 days and then tested at 37℃ on days 1, 3, 5, and 7.

[0017] In this paper, the sampling quantity for uniformity monitoring was 15 standard samples with the minimum packaging quantity. BCA protein quantification was performed twice per sample, and CD79A fluorescence quantitative PCR was performed three times per sample.

[0018] This invention also provides the application of the preparation method described above in the preparation of SPF porcine antibody-negative serum standard samples.

[0019] This invention also provides SPF porcine antibody-negative serum standard samples prepared by the method described above.

[0020] This invention also provides the application of the SPF porcine antibody-negative serum standard sample described above in the serological detection of swine diseases or the preparation of serological detection products for swine diseases, wherein the swine diseases include the following types: African swine fever, foot-and-mouth disease type O, classical swine fever, pseudorabies, porcine reproductive and respiratory syndrome, porcine circovirus type 2, transmissible gastroenteritis of swine, porcine epidemic diarrhea, swine rotavirus disease, and porcine parvovirus disease.

[0021] Compared with existing related technologies, it has the following characteristics and advantages: 1) Systematized screening process: A triple screening mechanism of "antibody initial screening - sterility test - ELISA and IIFA confirmation" is established, covering 10 common swine disease antibodies, effectively eliminating other common swine disease antibodies, cross-reaction and contamination risks, and the raw material specificity is significantly better than existing single screening methods.

[0022] 2) Standardization of preparation process: Clarify the ratio of freeze-drying protectant and freeze-drying procedure parameters to avoid quality fluctuations caused by inconsistent processes in traditional preparation processes and ensure consistency in batch production.

[0023] 3) Comprehensive quality assessment: For the first time, BCA assays for total protein and CD79A gene were used as targets and evaluation methods to conduct homogeneity, stability, and assessments on the prepared standard samples. Homogeneity testing employed dual-indicator detection of protein and nucleic acid and large-sample sampling. Stability assessment covered multiple scenarios, including repeated freeze-thaw cycles, short-term stability, and long-term stability. The assessment system complies with relevant national standards for the development of standard samples and is more scientific and reliable than existing single-indicator assessments.

[0024] 4) High practicality: The prepared standard samples can be directly used as quality control materials and negative controls for serological detection of various common swine diseases. They can be used for the research and development and quality control of test kits, as well as for kit comparison and proficiency testing. This fills the gap in standardized negative serum samples in this field in my country, helps to standardize the testing process and improve the comparability of test results. Attached Figure Description

[0025] Figure 1 The real-time fluorescence quantitative PCR amplification curve of the CD79A gene is shown.

[0026] Figure 2 The results of agarose gel electrophoresis of the CD79A gene conventional PCR amplification product are shown. Lanes 1-4 are, in order, the CD79A gene in serum of SD strain ASFV (positive control), the CD79 gene in negative serum, the CD79A gene in negative serum (repeat well), and the nucleic acid molecular weight standard (DL 2000 Marker). Detailed Implementation

[0027] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0029] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0030] The SPF pigs used in the following examples were purchased from the experimental animal base of the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0031] The following examples use statistical software to process the data. The experimental results are expressed as the coefficient of variation (CV). CV ≤ 5% indicates that the sample homogeneity / stability is good, and CV > 5% indicates that the sample homogeneity / stability does not meet the requirements.

[0032] Example 1: Preparation, identification, and quality assessment of standard serum samples negative for antibodies against common swine diseases. 1. Preparation and identification of raw materials for SPF pig negative serum standard samples Three healthy 40-day-old SPF pigs (provided by the Experimental Animal Base of the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences) were selected. Clinical examination confirmed the absence of abnormal symptoms such as fever and lethargy. A comprehensive test for antibodies against common swine diseases was performed using various ELISA kits, and all results were negative (Table 1). Information regarding the ELISA kits used is shown in Table 1. Other laboratory test results are for reference only; the ELISA test results described in this invention should be the core basis for judgment.

[0033] Table 1. Summary of Antibody Detection Results for Common Swine Diseases

[0034] 1) Preparation and testing of raw materials: Three qualified SPF pigs (40 days old) were euthanized, and all blood was aseptically collected from each pig. After standing at room temperature for 2 hours, the blood was centrifuged at 3000 rpm for 10 minutes at 4℃ to separate the serum. The serum from the three pigs was thoroughly mixed in equal volumes to obtain approximately 500 mL of mixed serum. An appropriate amount of the mixed serum was inoculated into TG medium (25℃, 37℃) and TSB medium (25℃), respectively. After culturing for 7 days under the indicated suitable temperature conditions, no colony growth was observed. Simultaneously, a mycoplasma detection kit was used for testing, and the results showed no mycoplasma growth. The results indicate that the standard sample raw material is pure and confirms sterility and no mycoplasma contamination.

[0035] 2) Inactivation and Inactivation Test: β-propiolactone was added to the serum to a final concentration of 0.1%, and the mixture was inactivated at 4°C for 72 hours. The inactivated virus solution was then inoculated into porcine alveolar macrophages (PAMs) at a concentration of 1% (v:v), and cultured at 37°C in a 5% CO2 incubator for 5 days. After harvesting the virus, it was subjected to one freeze-thaw cycle and then blindly passaged for 3 generations in PAMs. The sample from the last blind passage was subjected to IIFA to test for complete inactivation. A negative IIFA test result indicates that the raw material no longer contains infectious African swine fever virus.

[0036] 3) Preparation of standard samples: Mix the mixed serum with the lyophilization protectant (containing fetal bovine serum, cyclodextrin, etc.) at a volume ratio of 1:4, stir thoroughly, and then dispense 0.5 mL into lyophilization glass vials, preparing a total of 500 vials. Pre-freeze the dispensed samples at -80℃ for 2 hours, and then transfer them to a lyophilizer for lyophilization.

[0037] The mRNA level of CD79A gene in serum was detected by real-time PCR. The primers and probes used for real-time PCR were designed and synthesized in-house based on the CD79A sequence in NCBI (GenBank accession number: NM_001135962, updated April 2, 2025). The upstream primer F was 5'-CATGGATCTCTCAGAACTG-3' (SEQ ID No: 1), the downstream primer R was 5'-GTTGACTTCGCACCTGTAC-3' (SEQ ID No: 2), and the probe sequence was 5'-FAM-CATCCAGCAAGTGAACAAGAGCCA-BHQ1-3' (SEQ ID No: 3). The product length was 91 bp.

[0038] The product sequence is as follows: 5'-CATGGATCTCTCAGAACTGGGCCCCAAGGGCGAGCTGATCATCCAGCAAGTGAACAAGAGCCACAGGGCATGTACAGGTGCGAAGTCAAC-3' (SEQ ID No:4).

[0039] The amplification system consisted of 0.5 μL Go Script™ RT Mix, 10 μL Go Taq Probe qPCR Master Mix (2×), 0.6 μL upstream primer (10 pmol / μL), 0.6 μL downstream primer (10 pmol / μL), 0.6 μL probe (10 pmol / μL), 2.7 μL nuclease-free water, and 5 μL template. The reaction conditions were: reverse transcription at 45℃ for 30 min, pre-denaturation at 95℃ for 5 min, denaturation at 95℃ for 5 s, and annealing extension at 60℃ for 30 s (fluorescence was collected), for a total of 35 cycles.

[0040] The effectiveness of serum raw materials was confirmed by quantitative real-time PCR using CD79A: one serum sample was tested in triplicate, and the results showed that the CD79A gene was detected by real-time PCR. Ct The values ​​were 30.285, 29.296, and 29.763, with an average of 29.781, all within the effective amplification range, indicating good amplification stability and suitability as qualified raw materials for standard preparation (Table 2 and). Figure 1 ).

[0041] This embodiment includes a positive control for simultaneous detection. The positive control is a qualitative standard sample of positive serum for African swine fever virus strain SD / DY-I / 21. This standard sample is a standard sample developed in the National Standardization Management Committee's standard sample development plan project (project number S2023180) that was approved by our laboratory. For details, please refer to the "Notice of the National Standardization Management Committee on Issuing the Second Batch of National Standard Sample Development and Replication Plan Projects in 2023" (National Standardization Management Committee Document

[2023] No. 38). Positive control Ct The average value of the test results is 29.480. Table 2. Results of CD79A Quantitative PCR Detection

[0042] 2. Screening and identification of specific proteins in negative serum Using the negative serum standard samples obtained in step 1 as the research object, the total protein concentration of the three serum samples was determined by the BCA protein quantification kit (manufactured by Tiangen Biotech (Beijing) Co., Ltd., catalog number PA115-01). The results showed that the total protein concentrations were 2329.8 μg / mL, 2286.5 μg / mL, and 2310.2 μg / mL, respectively, with an average concentration of 2308.8 μg / mL.

[0043] Based on the results of CD79A gene fluorescence quantitative PCR detection, through CtValue analysis was used to verify the mRNA level of residual active B cells in serum, with CD79A mRNA as a specific monitoring indicator, providing a molecular marker basis for subsequent homogeneity and stability assessment of standard samples.

[0044] 3. Standard sample homogeneity testing and evaluation In accordance with the requirements of the "Standard Sample Working Guidelines Part 3: Standard Sample Value Determination and Homogeneity and Stability Assessment" (GB / T15000.3-2023), 15 standard samples with the minimum packaging quantity were selected using a random stratified sampling method.

[0045] Protein concentration in each sample was determined using the BCA protein quantification method (repeated twice) and the CD79A real-time PCR method. Ct The values ​​(repeated 3 times) were amplified strictly according to the established amplification system and reaction conditions.

[0046] Based on the BCA protein concentration determination and CD79A gene fluorescence quantitative PCR detection results, the detection mean and coefficient of variation (CV) of each sample were calculated. When the CV of all samples is <5% and the overall coefficient of variation of the 15 samples is <5%, the sample homogeneity is deemed to be qualified.

[0047] The results are shown in Table 3: the average protein concentration of the 15 samples was 940.837 μg / mL, and the overall coefficient of variation was 4.68%, indicating that the homogeneity between different unit bottles of the negative serum sample was qualified and the protein concentration detection index was qualified.

[0048] Table 3. Results of homogeneity test of antibody-negative serum standard samples (total protein concentration detected by BCA method)

[0049] The results of CD79A gene real-time PCR detection are shown in Table 4 below: The coefficient of variation for each sample ranged from 0.23% to 1.97%, all less than the 5% standard. The overall mean of the 15 samples was 29.701, with an overall coefficient of variation of 1.20%, which meets the requirements. Therefore, the samples showed good homogeneity and met the requirements for nucleic acid level detection.

[0050] Table 4. Results of homogeneity test of antibody-negative serum standard samples (CD79A gene detection by real-time PCR)

[0051] 4. Stability monitoring and evaluation of standard samples Repeated freeze-thaw stability assessment: Three standard samples were randomly selected and subjected to 0, 3, and 5 freeze-thaw cycles, respectively (freeze-thaw conditions: frozen at -20℃ for 2 hours, thawed at room temperature for 1 hour to fully thaw, constituting one freeze-thaw cycle). Protein concentration and stability were then detected using the BCA method and CD79A quantitative real-time PCR. Ct Values, calculate the coefficient of variation, and perform independent samples. t Test. When the coefficient of variation is <5% and the p-value is >0.05, the sample is deemed to have passed the test for repeated freeze-thaw stability.

[0052] The results of the post-treatment tests are shown in Tables 5 and 6. The coefficient of variation for protein concentration ranged from 0.00% to 4.35%, and the overall coefficient of variation was 1.61%. CD79A gene... Ct The coefficient of variation for the values ​​ranged from 0.15% to 1.43%, with an overall coefficient of variation of 0.55%, and the variation varied among different freeze-thaw groups. P The values ​​were all >0.05, indicating that the stability of the samples was qualified after repeated freeze-thaw cycles.

[0053] Table 5. Results of repeated freeze-thaw stability test of antibody-negative serum standard samples (protein concentration determined by BCA method)

[0054] Table 6. Results of repeated freeze-thaw stability test of antibody-negative serum standard samples (CD79A gene detected by real-time PCR)

[0055] Short-term stability assessment: Randomly selected negative serum standard samples were stored at -20℃, 4℃, 25℃, and 37℃. Testing was performed after 7 days at -20℃, 4℃, and 25℃, and on days 1, 3, 5, and 7 at 37℃. The relevant indicators were detected using the BCA method and CD79A quantitative PCR method, and the coefficient of variation was calculated. A CV < 5% was considered a satisfactory short-term stability.

[0056] The results are shown in Table 7. After storage at various temperatures, the average protein concentration was 886.89 μg / mL, with a coefficient of variation ranging from 0.00% to 4.99%, and an overall coefficient of variation of 3.74%. Similarly, the CD79A gene in African swine fever negative serum standard samples was detected by real-time PCR at different temperatures (37℃, 4℃, -20℃, 25℃) and at different days (days 1, 3, 5, and 7). The detection results were analyzed. Ct The values ​​were analyzed. The results are shown in Table 8, CD79A gene. CtThe average value was 29.068, the coefficient of variation ranged from 0.23% to 1.96%, and the overall coefficient of variation was 0.10%, all less than 5%. The results indicate that the short-term stability of the sample is satisfactory.

[0057] Table 7. Short-term stability test results of antibody and negative serum standard samples (protein concentration determined by BCA method)

[0058] Table 8. Short-term stability test results of antibody-negative serum standard samples (CD79A gene detection by quantitative real-time PCR)

[0059] In summary, using quantitative real-time PCR, the expression levels of BCA protein and the relative expression levels of the CD79A gene in antibody-negative serum standard samples from common swine diseases such as African swine fever were investigated under different temperatures (37℃, 4℃, -20℃, 25℃) and different days (days 1, 3, 5, and 7). Ct The antibody protein concentration and the CD79A gene fluorescence quantitative PCR value were detected and analyzed. The results showed that the antibody protein concentration of each sample and the values ​​obtained from the CD79A gene fluorescence quantitative PCR detection... Ct The coefficients of variation of the values ​​were all far below the 5% threshold, indicating that the overall coefficients of variation of the two indicators in all 21 samples were less than 5%, the dispersion of the test results among different samples was small, and the overall consistency of the test results of the samples was good.

[0060] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for preparing standard serum samples negative for antibodies against common specific pathogen (SPF) swine diseases, characterized in that, It includes the following steps: (1) Raw material preparation and initial screening: Select healthy SPF boars aged 40 days, collect blood aseptically and separate serum, and after heat inactivation treatment and inactivation test, use multiple enzyme-linked immunosorbent assay (ELISA) kits to detect serum antibodies against 10 swine diseases. The swine serum with negative test results is qualified SPF swine serum. (2) Serum testing: SPF pig serum that has passed the initial screening is subjected to sterility testing and mycoplasma testing to ensure that the serum is pure and uncontaminated; (3) Confirmation of negative serum: The mixed serum was repeatedly tested using ELISA and indirect immunofluorescence assay (IIFA) to confirm that all 10 swine disease antibodies were negative and had no cross-reactivity, thus obtaining negative serum raw materials; (4) Preparation of standard samples: The negative serum raw material is mixed with a freeze-drying protectant containing calf serum and cyclodextrin in an appropriate volume ratio and then freeze-dried. (5) Homogeneity test and evaluation: The total protein concentration was detected by BCA protein quantification kit and the CD79A gene mRNA level was detected by real-time PCR. The coefficient of variation (CV) was calculated. When the coefficient of variation CV < 5%, the sample homogeneity was deemed to be qualified. (6) Stability testing and evaluation: including repeated freeze-thaw stability, short-term stability and long-term stability testing and evaluation. When CV < 5%, the stability is deemed qualified and the negative serum standard sample of the common swine disease antibody is obtained.

2. The method according to claim 1, characterized in that, The 10 swine disease antibodies mentioned in step 1) are African swine fever virus (ASFV) antibody, foot-and-mouth disease virus type O (FMDV-O) antibody, classical swine fever virus (CSFV) antibody, pseudorabies virus (PRV) gB antibody, porcine reproductive and respiratory syndrome virus (PRRSV) antibody, porcine circovirus type 2 (PCV 2) antibody, transmissible gastroenteritis virus (TGEV) antibody, porcine epidemic diarrhea virus (PEDV) antibody, porcine rotavirus (PoRV) antibody, and porcine parvovirus (PPV) antibody.

3. The method according to claim 1, characterized in that, The aseptic processing steps described in step 2) are as follows: after mixing the separated serum, they are cultured in TG medium at 25℃ and 37℃ for 7 days respectively, and in TSB medium at 25℃ for 7 days. The presence of colony growth is observed daily. Then, mycoplasma testing is performed to confirm that there is no microbial contamination.

4. The method according to claim 1, characterized in that, The optimal ratio mentioned in step 4) is a volume ratio (v / v) of serum raw material to lyophilization protectant of 1:

4.

5. The method according to claim 1, characterized in that, The nucleotide sequence of the CD79A gene mentioned in step 5) is GenBank: NM_001135962, updated on April 2, 2025; the upstream primer sequence used for the real-time PCR is: 5'-CATGGATCTCTCAGAACTG-3'; the downstream primer sequence is: 5'-GTTGACTTCGCACCTGTAC-3'; and the probe sequence is: 5'-FAM-CATCCAGCAAGTGAACAAGAGCCA-BHQ1-3'.

6. The method according to any one of claims 1-5, characterized in that, The reaction system for quantitative real-time PCR described in step 5) includes 0.5 μL of Go Script™ RT Mix, 10 μL of Go Taq Probe qPCR Master Mix (2×), 0.6 μL of upstream primer (10 pmol / μL), 0.6 μL of downstream primer (10 pmol / μL), 0.6 μL of probe (10 pmol / μL), 2.7 μL of nuclease-free water, and 5 μL of template. The reaction conditions are: reverse transcription at 45℃ for 30 min, pre-denaturation at 95℃ for 5 min, followed by denaturation at 95℃ for 5 s and annealing extension at 60℃ for 30 s (collecting fluorescence), for a total of 35 cycles.

7. The method according to any one of claims 1-6, characterized in that, The repeated freeze-thaw stability assessment in step 6) is as follows: three standard samples are randomly selected and subjected to 0, 3, and 5 repeated freeze-thaw treatments. The nucleic acid (CD79A) level and total protein (BCA) level of each sample are repeatedly tested (BCA total protein test is repeated 2 times, and CD79A nucleic acid fluorescent PCR test is repeated 3 times). The short-term stability assessment is as follows: five samples are randomly selected and stored at -20℃, 4℃, 25℃, and 37℃ respectively. The samples are tested on the 7th day after storage at -20℃, 4℃, and 25℃, and on the 1st, 3rd, 5th, and 7th days after storage at 37℃.

8. The use of the method according to any one of claims 1-7 in the preparation of SPF porcine negative serum standard samples.

9. The SPF porcine antibody-negative serum standard sample prepared by the method according to any one of claims 1-7.

10. The application of the SPF porcine antibody-negative serum standard sample according to claim 9 in the serological detection of swine disease antibodies or the preparation of swine disease serological detection products, wherein the swine diseases include the following types: African swine fever, foot-and-mouth disease type O, classical swine fever, pseudorabies, porcine reproductive and respiratory syndrome, porcine circovirus type 2, transmissible gastroenteritis of swine, porcine epidemic diarrhea, swine rotavirus disease, and porcine parvovirus disease.

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