Method for preparing sheep-derived positive serum reference of yersinia enterocolitica, obtained positive serum and application thereof

Abstract

The invention discloses a method for preparing a sheep-derived positive serum reference of yersinia enterocolitica, obtained positive serum and application of the positive serum, and belongs to the field of veterinary medicine products. The invention aims to solve the technical problem of how to obtain the yersinia enterocolitica sheep-derived positive serum reference which is stable in titer and character and can be used for specific detection of brucella diagnostic products. Therefore, the invention provides a method for preparing the yersinia enterocolitica sheep-derived positive serum reference, which comprises the following steps: immunizing a tested sheep by using an immunizing antigen, and collecting serum of the immunized tested sheep to obtain the yersinia enterocolitica sheep-derived positive serum reference. The immune antigen is an inactivated yersinia enterocolitica obtained by inactivating the yersinia enterocolitica. The positive serum reference can be effectively applied to quality control evaluation work of brucellosis related diagnosis products, and plays a positive role in prevention and control of brucellosis.

Description

Technical Field

[0001] This application belongs to the field of veterinary pharmaceutical products, specifically relating to a method for preparing sheep-derived positive serum reference materials for Yersinia enterocolitica, the obtained positive serum and its application. Background Technology

[0002] Brucellosis is a zoonotic disease caused by Brucella infection, primarily resulting from the bacteria invading the mucous membranes and respiratory tract of the body. In livestock, infection mainly manifests as infertility and abortion in females, and orchitis in males. Detection of infected animals and evaluation of vaccine efficacy are crucial methods for brucellosis control. Ovine-derived Yersinia enterocolitica O9 serum is a key substance for evaluating the specificity of brucellosis diagnostic products. Currently, diagnostic product manufacturers prepare their own ovine-derived Yersinia enterocolitica O9 serum. Problems such as unclear bacterial strain sources, unclear animal backgrounds, and undetermined values ​​of immunoantigens and reactive antigens lead to inconsistent titers among these self-prepared sera, making them unsuitable as a physical benchmark for accurately evaluating the specificity of diagnostic products.

[0003] Based on the above issues, it is urgent to develop a sheep-derived positive serum reference material of Yersinia enterocolitica O9 that has a fixed titer and can evaluate the specificity of brucellosis diagnostic products, using animals with clean serum backgrounds, after systematic identification of the strains used in preparation. This would provide material support for accurately evaluating the quality of brucellosis diagnostic products, thereby improving the level of brucellosis prevention and control. Summary of the Invention

[0004] The technical problem to be solved by this invention is: how to obtain a sheep-derived positive serum reference for brucellosis diagnostic products that is stable in potency and properties and can be used for specific testing of brucellosis diagnostic products.

[0005] To solve this technical problem, this application provides the following technical solution: This invention provides a method for preparing a sheep-derived positive serum reference for Yersinia enterocolitica. The method may include immunizing test sheep with an immunogen, collecting serum from the immunized test sheep, and processing the serum to obtain the sheep-derived positive serum reference for Yersinia enterocolitica. The immunogen is inactivated Yersinia enterocolitica obtained by inactivating Yersinia enterocolitica.

[0006] In one specific embodiment of the present invention, the Yersinia enterocolitica is Yersinia enterocolitica O9, with the accession number CVCC 4444 at the China Veterinary Microbiology Culture Collection Center.

[0007] In this invention, the test sheep can be a 6-month-old goat.

[0008] In this invention, the test sheep also meet the following conditions: the results of brucellosis tube agglutination test, red benzene plate agglutination test, indirect ELISA, competitive ELISA, fluorescence polarization, Yersinia enterocolitica O9 tube agglutination, Salmonella Dublin tube agglutination, and Yersinia enterocolitica O9 tube agglutination test are all negative.

[0009] In this invention, the inactivation method can be high-temperature inactivation.

[0010] In this invention, the high-temperature inactivation method can be water bath heating.

[0011] In this invention, the temperature of the water bath heating can be 70 to 90°C.

[0012] In this invention, the water bath heating temperature can be 75 to 85°C.

[0013] In this invention, the water bath heating temperature can be 80°C.

[0014] In this invention, the water bath heating time can be from 60 minutes to 120 minutes.

[0015] In this invention, the water bath heating time can be 80 to 100 minutes.

[0016] In this invention, the water bath heating time can be 90 minutes.

[0017] In this invention, the water bath heating temperature can be 80°C, and the water bath heating time can be 90 minutes.

[0018] In this invention, the immunization method may be subcutaneous injection, and the number of immunizations may be at least three. The dose of the first subcutaneous injection is 1.2 × 10⁻⁶ based on the pre-inactivation Yersinia enterocolitica O9 agglutination assay. 11 CFU / animal / dose; the subcutaneous injection dose for revaccination is 2 × 10⁻⁶ based on the pre-inactivation Yersinia enterocolitica O9 agglutination assay. 11 CFU / animal / dose; the subcutaneous injection dose for the third immunization is 2 × 10⁻⁶ based on the pre-inactivation Yersinia enterocolitica O9 agglutination assay. 11 CFU / each / time.

[0019] In this invention, the interval between two consecutive immunizations can be 7 days.

[0020] In this invention, the method of collecting serum from immunized test sheep can be the collection of serum from immunized test sheep with a serum titer of 1:2000 or higher.

[0021] The collection of serum from immunized test sheep may include the steps of collecting blood from the test sheep with a serum titer of 1:2000 or higher after immunization and separating the serum to obtain sheep-derived positive serum candidates.

[0022] The blood collection can be performed according to conventional methods in the art.

[0023] The serum separation can be performed according to conventional methods in the art.

[0024] This invention also includes a step of sterility testing on the sheep-derived positive serum candidate. The sterility test can be performed in accordance with Appendix 3306 of the Chinese Veterinary Pharmacopoeia (2020 Edition).

[0025] This invention also includes a step of specifically detecting the sheep-derived positive serum candidates. The specific detection includes the brucellosis rose benzene plate agglutination test, the brucellosis tube agglutination test, the Escherichia coli O157 tube agglutination test, and the Salmonella Dublin tube agglutination test. In the specific detection, the brucellosis rose benzene plate agglutination titer is 1:10; the brucellosis tube agglutination titer is 1:12.5; the remaining tests are negative.

[0026] In this invention, the processing of the serum includes the steps of dilution and / or lyophilization.

[0027] In this invention, the dilution includes the step of diluting the sheep-derived positive serum candidate with negative serum.

[0028] The negative serum may be sheep negative serum, which may be derived from sheep that have not been immunized and whose results in the following tube agglutination tests are all negative: Brucellosis tube agglutination test, Rose Bengal plate agglutination test, Indirect ELISA, Competitive ELISA, Fluorescence polarization, Escherichia coli O157 tube agglutination, Salmonella Dublin tube agglutination, and Yersinia enterocolitica O9 tube agglutination test.

[0029] Preferably, the dilution factor can be 5 times.

[0030] In this invention, the freeze-drying parameters can be as follows: pre-freeze at -55°C for 3 h, vacuum for 30 min, raise the temperature to -16°C for 2 h and maintain for 11 h, raise the temperature to 26°C for 2 h and maintain for 6 h, and then end.

[0031] The present invention also provides a reference sample of sheep-derived positive serum of Yersinia enterocolitica O9 prepared by the above method.

[0032] The method provided by this invention prepares a sheep-derived positive serum reference for Yersinia enterocolitica O9, which may have at least one of the following characteristics: (1) Spongy, loose clumps that easily detach from the bottle wall and dissolve rapidly after adding sterile water; (2) Performed according to Appendix 3306 of the current Chinese Veterinary Pharmacopoeia, with sterile growth; (3) The serum titer was 1:100; (4) The tests were conducted according to Appendix 3204 of the current Chinese Veterinary Pharmacopoeia, and the remaining moisture content did not exceed 3.0%; (5) According to Appendix 3103 of the current Chinese Veterinary Pharmacopoeia, all samples showed a glow.

[0033] The present invention also provides a reagent and / or kit for evaluating the specificity of Brucella diagnostic products, wherein the reagent and / or kit may contain the above-mentioned Yersinia enterocolitica sheep-derived positive serum reference.

[0034] The present invention also provides a method for evaluating the specificity of Brucella diagnostic products, the method comprising the steps of evaluating the specificity of Brucella diagnostic products using the above-described Yersinia enterocolitica sheep-derived positive serum reference and / or the above-described reagents and / or kits.

[0035] This invention also provides the use of the above-mentioned sheep-derived positive serum reference material for Yersinia enterocolitica in at least one of the following: A1) Application in detecting or assisting in the detection of the specificity of Brucella diagnostic products; A2) Application in the preparation of products for the specificity of diagnostic products for detecting or assisting in the detection of Brucella; A3) Application in evaluating or assisting in the evaluation of the specificity of Brucella diagnostic products; A4) Application in the preparation of products for evaluating or assisting in the evaluation of the specificity of Brucella diagnostic products; A5) Application in the detection or auxiliary detection of Yersinia enterocolitica; A6) Application in the preparation of products for the detection or auxiliary detection of Yersinia enterocolitica.

[0036] In this invention, the product may be a reagent or a kit. The reagent or kit can be used for the specific detection of Brucella diagnostic products.

[0037] The present invention also provides the use of the above-described reagents and / or kits in at least one of the following: B1) Application in detecting or assisting in the detection of the specificity of Brucella diagnostic products; B2) Application in evaluating or assisting in the evaluation of the specificity of Brucella diagnostic products; B3) Application in the detection or auxiliary detection of Yersinia enterocolitica.

[0038] The beneficial effects of this invention are: the reference strain of sheep-derived positive serum uses inactivated antigens prepared from systematically identified Yersinia enterocolitica type O9, and the antigen concentration is calibrated to 4 × 10⁻⁶. 10 CFU / mL, 6-month-old sheep with a clean serum background were intravenously vaccinated with three doses of 3 mL / head, 5 mL / head, and 5 mL / head, 7 days apart. Seven days after the third immunization, the titer was determined by blood testing. Sheep serum with an agglutination titer of not less than 1:2000 was collected in test tubes and subjected to semi-finished product testing before lyophilization. After a series of processes including finished product testing, uniformity testing, and titer calibration, it became a sheep-derived positive serum reference material. The sheep-derived positive serum reference material has a short preparation cycle and a fixed titer of 1:100. Its titer was determined using a reaction antigen concentration calibrated to the same turbidity and OD value as the national reference material for Brucella tube agglutination test antigen diluted 20 times. 600 nm =0.8 to 0.9. This positive serum reference material is used in the quality control evaluation of brucellosis-related diagnostic products, playing a positive role in the prevention and control of brucellosis. Attached Figure Description

[0039] Figure 1 The results show the culture characteristics of Yersinia enterocolitica on TSA agar plates.

[0040] Figure 2 Gram staining results for Yersinia enterocolitica.

[0041] Figure 3 The results of serum agglutination reaction between Yersinia enterocolitica and Yersinia enterocolitica O9 single factor (left is antigen control, right is reaction).

[0042] Figure 4 GO functional classification diagram for gene function annotation of Yersinia enterocolitica.

[0043] Figure 5 KEGG functional classification diagram for gene function annotation of Yersinia enterocolitica.

[0044] Figure 6 The results are from the serum titer assay in Laboratory 1.

[0045] Figure 7 The results are from the serum titer assay in Laboratory 2. Detailed Implementation

[0046] I. Terminology in this application Examples of resources describing many of the molecular biology-related terms used in this article can be found in the following literature: Alberts et al., Molecular Biology of The Cell, 5th ed., Garland Science Publishing, Inc.: New York, 2007; Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed., Springer-Verlag: New York, 1991; King et al., A Dictionary of Genetics, 6th ed., Oxford University Press: New York, 2002; and Lewin, GenesIX, Oxford University Press: New York, 2007.

[0047] Any references cited in this article, including, for example, all patents, published patent applications and non-patent publications, are incorporated in their entirety by reference.

[0048] For ease of understanding of this disclosure, several terms and abbreviations used herein are defined as follows: When used in a list of two or more items, the term "and / or" means that any of the listed items can be used alone or in combination with any one or more of the listed items. For example, the expression "A and / or B" is intended to mean either or both of A and B, i.e., A alone, B alone, or a combination of A and B. The expression "A, B and / or C" means A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C.

[0049] II. Implementation Examples The present application will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the present application and are 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 present application in any way.

[0050] 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.

[0051] In the following embodiments, the Yersinia enterocolitica strain is accessed at the China Veterinary Microbiological Culture Collection Center with the accession number CVCC 4444. Unless otherwise specified, in the following embodiments, Yersinia enterocolitica, Yersinia enterocolitica O9, and Yersinia enterocolitica CVCC4444 have the same meaning.

[0052] The methods for the brucellosis tube agglutination test and the brucellosis rose benzene plate agglutination test were performed in accordance with the national standard "Diagnostic Techniques for Animal Brucellosis" (standard number: GB / T 18646-2018). The reagents used include: the national reference material for brucellosis tube agglutination test antigen, CVCC number Z11, batch number 20230715 (the website of the National Veterinary Microbiology Culture Collection Center (CVCC) is https: / / syjg.agri.cn / sso-client-oauth2 / shopping / index?keywords=99999&type=2# / shopping); the national standard material for brucellosis positive serum, CVCC number Z291, batch number 202301; the reference material for brucellosis negative serum (sheep-derived), CVCC number Z394, batch number 202301; and the national reference material for brucellosis rose benzene plate agglutination test antigen, CVCC number Z6, batch number 202301. The public can purchase or apply for the relevant materials from the National Veterinary Microbiology Culture Collection Center. Brucellosis indirect ELISA and brucellosis competitive ELISA assays were performed using the following kits according to the product instructions: Brucellosis Competitive ELISA Antibody Detection Kit, batch number Bru230315, purchased from Guangzhou Yueyang Biotechnology Co., Ltd.; and Brucellosis Fluorescence Polarization Kit, batch number S20230316Y, purchased from Zhonglianrui (Beijing) Biotechnology Co., Ltd. Escherichia coli O157 tube agglutination assay, Salmonella Dublin tube agglutination assay, and Yersinia enterocolitica O9 tube agglutination assay were performed according to the tube agglutination assay method.

[0053] In the following examples, the Proclin 300 used for corrosion protection is a Merck product with part number 48915-U.

[0054] In the following examples, the TSA agar plate culture medium was formulated as follows: 15.0 g tryptone, 5.0 g soybean peptone, 5.0 g sodium chloride, 15.0 g agar, 1000 mL distilled water, and pH 7.3 ± 0.2 (25 °C).

[0055] The TSB medium formula is 17 g peptone, 3 g yeast extract, 2.5 g glucose, and 5 g NaCl. Dissolve the above ingredients (and 15.0 g agar if in solid form) in 1000 mL of distilled water and adjust the pH to 7.0.

[0056] Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0057] Example 1: Preparation and testing of sheep-derived positive serum for Yersinia enterocolitica. I. Identification of strains used in preparation 1. Cultivation characteristics The bacterial strain used for preparation was *Yersinia enterocolitica* serotype O9, which was reconstituted with physiological saline and inoculated into TSB medium at 25°C for 48 hours, resulting in a uniformly turbid culture. The culture was then inoculated onto TSA medium and incubated at 25°C for 48 hours, and the bacterial culture characteristics were observed and recorded. The strain formed translucent milky-white colonies on TSA medium. Figure 1 .

[0058] 2. Morphological and biochemical characteristics Freshly cultured Yersinia enterocolitica from TSA medium was subjected to Gram staining. Bacterial morphology was observed and recorded. The bacteria were Gram-negative, oval or short rod-shaped. Figure 2 Biochemical identification showed urease positive, hydrogen sulfide negative, VP25℃ positive, and ornithine decarboxylase positive. It ferments glucose and sucrose to produce acid but not gas, and does not ferment disaccharide or rhamnose, consistent with the characteristics of Yersinia enterocolitica in bacterial taxonomy.

[0059] 3. Serological characteristics After reconstitution with 0.5 mL of physiological saline, the culture was inoculated into TSB and cultured at 25°C for 48 h; then inoculated into TSA slant tubes and cultured at 25°C for 48 h; finally, it was subcultured and inoculated into TSA slant tubes and cultured at 25°C for 48 h. 0.5% phenol-saline (3 mL / tube) was added to the slant culture in the tubes, the bacterial growth was washed off, and the culture was autoclaved at 121°C for 1 h. Agar agglutination was then performed with Yersinia enterocolitica O9 single-factor serum, and the agglutination was recorded. Figure 3 .

[0060] 4.16S rDNA PCR detection of 16S rDNA: The upstream primer for PCR amplification was 5′-AGA GTT TGA TCC TGG CTC AG (SEQ ID NO:1)-3′, and the downstream primer was 5′-ACG GCT ACC TTG TTA CGA CTT (SEQ ID NO:2)-3′. The electrophoretic band of the Yersinia enterocolitica 16S rDNA PCR product was 1505 bp, consistent with expectations. Nucleic acid sequence alignment confirmed it as Yersinia enterocolitica.

[0061] 5. Whole genome sequencing Genomic DNA was extracted using the Tianlong fully automated bacterial DNA kit. After agarose gel electrophoresis, quantification was performed using Qubit. The genome of Yersinia enterocolitica O9 was sequenced using the MGISEQ-200 platform from BGI Genomics. Gene function was predicted using two databases: GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes).

[0062] (1) Sequencing and Assembly Results: Based on the assembled genome sequence of *Yersinia enterocolitica* strain and the predicted results of coding genes, the *Yersinia enterocolitica* genome was displayed using MGAP V2.0.0 software, and the gene function annotation analysis results were also presented. The results showed that the *Yersinia enterocolitica* genome size was 4540172 bp, and the G+C content was 47.11%. The outermost ring of the Yersinia enterocolitica genome circular diagram shows the genome sequence location coordinates. From the outside in, these represent: GC content distribution; read depth statistics (depth values ​​converted to logarithmic form); genome functional regions, mainly showing the distribution of CDS, rRNA, and tRNA on the positive and reverse strands; SNP density distribution heatmap (the darker the color, the more SNP sites in the region); Indel density distribution heatmap (the darker the color, the more Indel sites in the region); CNV distribution regions (dark blue indicates regions with deletion detection, orange indicates regions with duplication detection); SV distribution (red indicates fragment insertion regions, green indicates fragment deletion regions); and SV variants including inversions (INV), intrachromosomal translocations (ITX), and interchromosomal translocations (CTX).

[0063] The outermost circle shows the coordinates of the genome sequence location. From the outside in, these represent: the distribution of GC content in the genome; read depth statistics, with depth values ​​converted to logarithmic form; functional regions of the genome, mainly showing the distribution of CDS, rRNA, and tRNA on the positive and reverse strands; a heatmap of SNP density distribution, with darker colors indicating a higher number of SNP sites in the region; a heatmap of Indel density distribution, with darker colors indicating a higher number of Indel sites in the region; CNV distribution regions, with dark blue indicating regions where deletions are detected and orange indicating regions where duplications are detected; SV distribution, with red indicating fragment insertion regions and green indicating fragment deletion regions; and SV variants including inversions (INV), intrachromosomal translocations (ITX), and interchromosomal translocations (CTX).

[0064] (2) Genomic annotation results matched 3132 genes of Yersinia enterocolitica serotype O:9 to GO analysis. Compared to molecular functions and biological processes, cellular components contain the most genes, mainly used to describe subcellular structures, locations, and macromolecular complexes, such as nucleoli, telomeres, and recognition initiation complexes. Cellular anatomical entities and membrane-related entities are particularly prominent. See Figure 4 . Figure 4 The horizontal axis represents the next level of GO terms in the three major GO categories, and the vertical axis represents the number of genes annotated under that term (including its subterms). The three different classifications represent the three basic categories of GO terms (from left to right: biological processes, cellular components, and molecular functions).

[0065] KEGG analysis showed that the KEGG pathway annotations of Yersinia enterocolitica serotype O:9 were mainly distributed in metabolic pathways, biosynthesis of secondary metabolites, biosynthesis of antibiotics, microbial metabolism in diverse environments, biofilm formation - Escherichia coli, carbon metabolism, biosynthesis of amino acids, nucleotide excision repair, aminoacyl - tRNA biosynthesis, and two - component system, as shown in Figure 5 . Figure 5 The numbers on the bar graph represent the number of annotated genes; the other coordinate axis is the code of each functional class at level 1 in the database. The explanations of the codes are shown in the corresponding legend.

[0066] II. Preparation and testing of immune antigens and reactive antigens 1. Preparation and testing of immune antigens (1) Preparation of immune antigens Inoculate Yersinia enterocolitica by streaking on TSA agar plate medium and culture at 25 °C for 48 h. Pick a single colony and streak it densely on a TSA agar slant, then culture at 25 °C for 48 h. Wash the bacterial lawn with normal saline to make a bacterial suspension. Conduct sterility test according to Appendix 3306 of the current Chinese Veterinary Pharmacopoeia, and immediately count the viable bacteria of the prepared bacterial suspension. According to the counting result, concentrate it to prepare a bacterial suspension of 4×10 10 CFU / mL, and inactivate it in a water bath at 80 °C for 90 min as the immune antigen.

[0067] (2) Testing of immune antigens Sterility test was carried out according to Appendix 3306 of the current Chinese Veterinary Pharmacopoeia and passed.

[0068] 2. Preparation and testing of reactive antigens (1) Preparation of reactive antigens Mix 1 mL of the prepared immunizing antigen with different amounts (0.5 mL, 0.6 mL, 0.7 mL, and so on) of 0.5% phenol 10% sodium chloride solution to obtain a mixed solution and perform OD 600 nm measurement to determine the mixing ratio when the OD 600 nm value is between 0.8 and 0.9 and record it. Then mix the remaining antigen solution with 0.5% phenol (5 g / L) 10% sodium chloride solution according to this mixing ratio to prepare the reaction antigen for the Yersinia enterocolitica O9 tube agglutination test.

[0069] (2) Inspection of the reaction antigen The OD 600 nm value for antigen concentration measurement is 0.813.

[0070] Sterility inspection is carried out according to Appendix 3306 of the current "Chinese Veterinary Pharmacopoeia" and is qualified.

[0071] 3. Preparation of turbidity tubes Dilute the prepared reaction antigen for the Yersinia enterocolitica O9 tube agglutination test by 2 times and mix it with 0.5% phenol 10% sodium chloride solution according to the ratio in Table 1 to prepare turbidity tubes. Obvious differences should be visible by naked eye observation between each turbidity tube.

[0072] Table 1 Preparation method of turbidity tubes for the Yersinia enterocolitica tube agglutination test

[0073] III. Preparation and inspection of sheep-derived positive serum 1. Preparation of sheep-derived positive serum (1) Screening of experimental animals Select 6-month-old goats that are negative in the Brucella tube agglutination test, Rose Bengal plate agglutination test, indirect ELISA, competitive ELISA, fluorescence polarization, Escherichia coli O157 tube agglutination, Salmonella dublin tube agglutination, and Yersinia enterocolitica O9 tube agglutination tests.

[0074] Among them, there are a total of 6 test sheep, their breed is goat, their weight is between 40 kg and 50 kg, and their source is Beijing Fulong Tengfei Laboratory Animal Research Institute Co., Ltd.

[0075] (2) Immunization program Intravenous injection of 3 mL of the immunizing antigen to the test sheep (calculated by Yersinia enterocolitica O9 before inactivation, the immunization dose is 1.2×10 11 CFU / sheep). On the day of the first immunization, it is recorded as the first day of the first immunization. Seven days after the first immunization, perform the second immunization, and intravenously inject 5 mL of the immunizing antigen (calculated by Yersinia enterocolitica O9 before inactivation, the immunization dose is 2×10 11CFU / animal), followed by a third immunization 7 days later, with an intravenous injection of 5 mL of immunogen (calculated as 2 × 10⁻⁶ Yersinia enterocolitica O9 before inactivation, immunization dose is 2 × 10⁻⁶). 11 CFU / animal), blood was collected 7 days later to determine serum titer. The serum titer should not be lower than 1:2000 (if the serum titer is lower than 1:2000, immunization can be boosted, and blood can be collected again after the titer reaches 1:2000). A large amount of blood was collected, the serum was separated and filtered and sterilized using a 0.22μm filter, and used as a candidate for sheep-derived positive serum of Yersinia enterocolitica O9.

[0076] 2. Detection and freeze-drying of positive serum candidates from sheep (1) Sterility test shall be performed in accordance with Appendix 3306 of the current Chinese Veterinary Pharmacopoeia, and sterile growth shall be observed.

[0077] (2) Potency determination: Sheep-derived positive serum was reconstituted with 1 mL of sterile water and then diluted with 0.5% phenol and 10% sodium chloride solution at ratios of 1:12.5, 1:25, 1:50, 1:75, and 1:100. 0.5 mL of each diluted serum was then mixed with an equal volume of Yersinia enterocolitica O9 reaction antigen (prepared in Part II, “2. Preparation and Testing of Reaction Antigen”) for agglutination tests. The final serum dilutions after adding the antigen were considered to be 1:25, 1:50, 1:100, 1:150, and 1:200, respectively. The solutions were incubated at 37°C for 24 hours, and the agglutination results were observed using a control turbidimetric tube. A final 1:100 dilution of sheep-derived positive serum showed a gglutination level of “++” or higher, while a final 1:150 dilution showed a gglutination level of “+” or “-”.

[0078] (3) Specificity tests: Brucella agglutination test, Brucella tube agglutination test, Escherichia coli O157 tube agglutination test, and Salmonella Dublin tube agglutination test were performed on the positive serum candidates from sheep. The agglutination titer of Brucella agglutination test was 1:10; the agglutination titer of Brucella tube agglutination test was 1:12.5; and the other tests were negative.

[0079] (4) Lyophilization of sheep-derived positive serum: The sheep-derived positive serum was diluted 5-fold using Brucella-negative serum (sheep-derived) reference material (CVCC number Z394, batch number 202301) according to the selected dilution. 0.05% Proclin 300 was added to the total volume of the diluted serum for preservation. 1 mL was added to each vial using a calibrated pipette, and the solution was lyophilized under vacuum. The lyophilizer was a SIM PiloFD8-2001V. The lyophilization program was as follows: pre-freeze at -50℃ for 2 hours, sublimate once to -16℃ for 15 hours, sublimate twice to 26℃ for 8 hours, and then the process was completed. Lyophilized Yersinia enterocolitica O9 sheep-derived positive serum reference material was obtained.

[0080] Equal volumes of freeze-dried sheep-derived positive serum containing Yersinia enterocolitica were prepared.

[0081] 3. Positive serum test for sheep-derived blood (1) Homogeneity test: The three positive sheep sera were reconstituted with 1 mL of sterile water and their potency was determined. The potency of the three positive sheep sera was 1:100.

[0082] (2) Collaborative Calibration: Twelve bottles of sheep-derived positive serum were randomly selected and submitted to two CNAS-accredited laboratories for collaborative titer calibration. Two lab technicians from each institution performed titer determination on three bottles of sheep-derived positive serum. The results of titer determinations by different technicians at different times and in different laboratories were statistically analyzed to calibrate the serum titer. The two technicians from Laboratory 1 performed titer determinations on three bottles of sheep-derived positive serum according to 3.2.3. The titer results for all three bottles were 1:100. (See...) Figure 7 Two inspectors in Laboratory 2 performed titer tests on three bottles of sheep-derived positive serum according to procedure 3.2.3, and the titer results for all three bottles were 1:100. Based on the titer test results of 12 bottles of sheep-derived positive serum by four inspectors from the two laboratories, the titer of this batch of sheep-derived positive serum was calibrated to 1:100.

[0083] (3) Effect of different preservation conditions on the titer of sheep-derived positive serum Experimental method: 12 bottles of sheep-derived positive serum were extracted and placed in 37℃, 25℃, 2 to 8℃ and -20℃ conditions, 3 bottles of each condition. At 7 days, 14 days, 21 days and 28 days of storage, 1 bottle of sheep-derived positive serum was taken from each storage condition for titer determination to analyze the stability of sheep-derived positive serum under different storage conditions.

[0084] The titers of sheep-derived positive serum stored at 37℃ were 1:100, 1:100, 1:50, and 1:50 after 7, 14, 21, and 28 days, respectively.

[0085] The titers of sheep-derived positive serum stored at 25℃ were 1:100, 1:100, 1:50, and 1:50 after 7, 14, 21, and 28 days, respectively.

[0086] The titers of sheep-derived positive serum stored at 2 to 8℃ were 1:100, 1:100, 1:100, and 1:100, respectively, after 7, 14, 21, and 28 days.

[0087] The titers of sheep-derived positive serum stored at -20℃ were 1:100, 1:100, 1:100, and 1:100 after 7, 14, 21, and 28 days, respectively.

[0088] Based on the experimental results regarding the impact of different storage conditions on the titer of sheep-derived positive serum, the optimal storage condition for sheep-derived positive serum was determined to be -20℃. The titer of sheep-derived positive serum stored at -20℃ was then periodically monitored. During serum transportation, ice packs should be used to ensure that the transportation temperature does not exceed 8℃, and the transportation time should ideally be controlled within 14 days.

[0089] 4. Finished product inspection (1) Properties: Spongy, loose clumps that easily separate from the bottle wall and dissolve rapidly after adding sterile water.

[0090] (2) Sterility test shall be performed in accordance with Appendix 3306 of the current Chinese Veterinary Pharmacopoeia, and sterile growth shall be observed.

[0091] (3) Potency determination: Sheep-derived positive serum was diluted 1:12.5, 25, 50, 75, and 100 times with 0.5% phenol and 10% sodium chloride solution, and then reacted with Escherichia coli O157 agglutination test antigen (OD). 600 nm For serum with agglutination values ​​between 0.8 and 0.9, a tube agglutination test was performed. After adding antigen, the serum dilutions became 1:25, 50, 100, 150, and 200. The tubes were incubated at 37°C for 24 hours, and turbidity was compared using a turbidimetric tube. The highest serum dilution showing agglutination of "++" or higher was determined as the serum titer. The final 1:100 dilution of sheep-derived positive serum showed agglutination of "++", and the final 1:150 dilution showed agglutination of "+". The serum titer was 1:100.

[0092] (4) The remaining moisture content was determined according to Appendix 3204 of the current Chinese Pharmacopoeia, and the remaining moisture content did not exceed 3.0%.

[0093] (5) The vacuum degree was measured according to Appendix 3103 of the current Chinese Veterinary Pharmacopoeia, and all showed a glow.

[0094] Example 2: Application of sheep-derived positive serum reference material for Yersinia enterocolitica O9 in evaluating the specificity of a brucellosis diagnostic kit. The specificity evaluation of brucellosis diagnostic products was carried out using a sheep-derived positive serum reference of *Yersinia enterocolitica* O9, which was reconstituted with 1 mL of sterile water as the test serum. This serum was used to evaluate the specificity of brucellosis diagnostic products, including the Brucella competitive ELISA antibody detection kit (published in Announcement No. 2356 of the Ministry of Agriculture of the People's Republic of China), the Brucella antibody test strip (published in Announcement No. 2526 of the Ministry of Agriculture of the People's Republic of China), the Brucella fluorescent polarization antibody detection kit (Announcement No. 471 of the Ministry of Agriculture and Rural Affairs), and the sheep Brucella indirect ELISA antibody detection kit (Announcement No. 604 of the Ministry of Agriculture and Rural Affairs of the People's Republic of China). The detection methods were performed according to the instructions of the above-mentioned diagnostic products, and the results were all negative.

[0095] The above results indicate that the sheep-derived positive serum reference material of Yersinia enterocolitica O9 prepared in this invention can be used for the specificity evaluation of brucellosis diagnostic products.

[0096] Based on a comprehensive and systematic identification of the bacterial strains used for serum preparation, including their culture characteristics, morphological and biochemical characteristics, serological characteristics, 16S rDNA PCR detection, and whole-genome sequencing, preparation processes for reactive antigens used for serum titer calibration and immunoantigens used for serum preparation were established, and the concentration of the immunoantigens was determined to be 4 × 10⁻⁶. 10 CFU / mL, the concentration of the reactive antigen was determined to be consistent with the reference concentration of the Brucella tube agglutination test antigen diluted 20-fold (OD). 600 nm (Values ​​between 0.8 and 0.9); animal screening standards and serum preparation processes for sheep-derived positive serum were established. After the sheep-derived positive serum titer was calibrated to 1:100, it was vacuum freeze-dried. The freeze-dried serum underwent comprehensive testing, including appearance, sterility, titer determination, homogeneity, residual moisture determination, and vacuum degree determination. The serum titer was calibrated by four inspectors from two CNAS-accredited laboratories, and the final serum titer was calibrated to 1:100. Experiments were conducted on the effects of different storage conditions on the titer of sheep-derived positive serum. The results showed that the suitable storage temperature for sheep-derived positive serum was -20℃, and cold chain transportation was recommended. The transportation temperature should not exceed 8℃, and the transportation time should be controlled within 14 days.

[0097] The present application has been described in detail above. Those skilled in the art will recognize that the present application can be implemented in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments are given in this application, it should be understood that further modifications can be made to the present application. In summary, in accordance with the principles of this application, this application is intended to include any changes, uses, or improvements to the present application, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for preparing sheep-derived positive serum reference material for Yersinia enterocolitica, characterized in that: The method includes immunizing test sheep with an immunogen, collecting serum from the immunized test sheep, and processing the serum to obtain a sheep-derived positive serum reference for Yersinia enterocolitica. The immunogen is an inactivated Yersinia enterocolitica obtained by inactivating Yersinia enterocolitica.

2. The method according to claim 1, characterized in that: The test sheep were 6-month-old goats.

3. The method according to claim 1 or 2, characterized in that: The immunization is administered intravenously, and the number of immunizations is at least three. The dose of the first intravenous immunization is 1.2 × 10⁻⁶ based on the pre-inactivation Yersinia enterocolitica agglutination assay. 11 CFU / animal / dose; the intravenous dose for revaccination is 2 × 10⁻⁶ based on the pre-inactivated Yersinia enterocolitica tube agglutination assay. 11 CFU / animal / dose; the intravenous dose for the third immunization is 2 × 10⁻⁶ based on the pre-inactivated Yersinia enterocolitica tube agglutination assay. 11 CFU / each / time.

4. The method according to claim 1, characterized in that: The inactivation is performed at high temperature.

5. The method according to claim 4, characterized in that: The high-temperature inactivation temperature is 80°C, and the high-temperature treatment time is 90 minutes.

6. The sheep-derived positive serum reference material for Yersinia enterocolitica prepared by the method according to any one of claims 1-5.

7. A reagent and / or kit for evaluating the specificity of Brucella diagnostic products, characterized in that: The reagents and / or kits contain the sheep-derived positive serum reference material for Yersinia enterocolitica as described in claim 6.

8. A method for evaluating the specificity of Brucella diagnostic products, characterized in that: The method includes the step of evaluating the specificity of Brucella diagnostic products using the sheep-derived positive serum reference of Yersinia enterocolitica as described in claim 6 and / or the reagents and / or kits as described in claim 7.

9. The use of the sheep-derived positive serum reference material for Yersinia enterocolitica as described in claim 6 in at least one of the following: A1) Application in detecting or assisting in the detection of the specificity of Brucella diagnostic products; A2) Application in the preparation of products for the specificity of diagnostic products for detecting or assisting in the detection of Brucella; A3) Application in evaluating or assisting in the evaluation of the specificity of Brucella diagnostic products; A4) Application in the preparation of products for evaluating or assisting in the evaluation of the specificity of Brucella diagnostic products; A5) Application in the detection or auxiliary detection of Yersinia enterocolitica; A6) Application in the preparation of products for the detection or auxiliary detection of Yersinia enterocolitica.

10. The use of the reagent and / or kit according to claim 7 in at least one of the following: B1) Application in detecting or assisting in the detection of the specificity of Brucella diagnostic products; B2) Application in evaluating or assisting in the evaluation of the specificity of Brucella diagnostic products; B3) Application in the detection or auxiliary detection of Yersinia enterocolitica.

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