WO2021227514A1 - 惰性载体间接凝集试验检测系统及其应用 - Google Patents

惰性载体间接凝集试验检测系统及其应用 Download PDF

Info

Publication number
WO2021227514A1
WO2021227514A1 PCT/CN2020/140030 CN2020140030W WO2021227514A1 WO 2021227514 A1 WO2021227514 A1 WO 2021227514A1 CN 2020140030 W CN2020140030 W CN 2020140030W WO 2021227514 A1 WO2021227514 A1 WO 2021227514A1
Authority
WO
WIPO (PCT)
Prior art keywords
detection system
inert carrier
salmonella
pullorum
agglutination test
Prior art date
Application number
PCT/CN2020/140030
Other languages
English (en)
French (fr)
Inventor
朱国强
杨斌
羊扬
孟霞
夏芃芃
段强德
朱晓芳
Original Assignee
扬州大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 扬州大学 filed Critical 扬州大学
Priority to GB2201199.3A priority Critical patent/GB2600848B/en
Publication of WO2021227514A1 publication Critical patent/WO2021227514A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/255—Salmonella (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/255—Salmonella (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells

Definitions

  • the invention belongs to the technical field of biomedicine and immunodiagnostic detection, and specifically relates to an inert carrier indirect agglutination test detection system.
  • the indirect agglutination test detection system includes the expression of the inert carrier Salmonella S9 and the new inert carrier indirect agglutination test pullorum and/or Salmonella typhoid detection system S9-P established with a single antigen factor P.
  • Pullorum is a bacterial infectious disease of chickens, turkeys and other poultry caused by Salmonella pullorum (Salmonella pullorum) infection. It mainly harms chicks within three weeks of age and has a high mortality rate. Adult chickens have no obvious symptoms after infection, but they can be sourced from breeding eggs. Vertical transmission to offspring of chicks is an important disease that harms the chicken industry in my country. Salmonella gallinarum (Salmonella gallinarum) infection causes bacterial infectious diseases in poultry such as chickens and turkeys. It is also an important disease that harms the chicken industry in my country. Pullorum and Salmonella typhi are classified as Class II animal diseases. Pullorum is also one of the animal diseases that must be notified by the World Organization for Animal Health (OIE).
  • OIE World Organization for Animal Health
  • the principle is that after the bacterial granular diagnostic antigen is combined with the corresponding serum antibody, when the electrolyte is present and the temperature is appropriate, the antigen particles and the corresponding specific antibody will be combined, and mutual agglutination will occur, forming agglomerated small pieces or particles, which only need to pass the naked eye. You can observe and judge the results on the spot.
  • the phenomenon of granular agglutination is a positive agglutination reaction.
  • the agglutination test is suitable for the rapid diagnosis of pathogenic infection and has many advantages, such as: simple and fast, does not require any additional (laboratory) equipment, is low-cost, and can be tested on-site with only a drop of chicken blood in the enclosure.
  • the agglutination antigen test has been in practice for many years, it has gradually exposed its limitations and technical bottlenecks in practical applications, such as the most widely used domestic commercial pullorum and typhoid agglutination antigen detection and diagnosis.
  • agglutinating antigen is composed of a reference strain of Salmonella pullorum and (or) a variant strain, which is a whole bacterial antigen of a mixture of different strains.
  • this multi-component antigen composite component that exists in the whole bacteria, many components may have non-specific crossover with Enterobacteriaceae in the same family, genus, and species, other families, genera and species of bacteria and other components including heterophilic antigens.
  • Reaction in view of the high working concentration and bacterial count required for the preparation of agglutinating antigen (Salmonella pullorum agglutinating antigen concentration has reached the order of 10 10 cfu bacteria), the disadvantages of non-specific cross-reaction caused by high antigen concentration will inevitably affect or even significantly interfere with monitoring , The accuracy of testing and diagnosis results.
  • the antigenic composition of the whole bacteria of the mixed bacteria is complex, and the antigenic components exposed on the surface of the bacteria are diverse and not single.
  • Many bacteria have common antigens or very close antigenicities with pullorum and Salmonella gallisepticum, including Escherichia coli, micrococcus and streptococcus (especially It is Gram D streptococcus) that can infect poultry.
  • pullorum and Salmonella gallisepticum including Escherichia coli, micrococcus and streptococcus (especially It is Gram D streptococcus) that can infect poultry.
  • a positive result in fact, also includes Staphylococcus epidermidis, Aerobacter, Proteus and certain species of Alexandria, Providenella and Citrobacter, etc., which are non-specific in the whole blood of chickens
  • the cross-reaction can range from a few chickens to 30%-40%; at the same time, considering the three components of bacterial antigens O 1 , O 9 , and O 12 , its oligosaccharide antigenicity is weak, of which O 12 also has 3 variants. That is, there are standard, variant, and intermediate types of Salmonella pullorum, resulting in poor specific response and poor matching between the diagnostic antigen strain and the infected serum.
  • the O antigen has a spatial conformation on the surface of the bacteria.
  • the impact and display are limited, leading to the low sensitivity of this method, and there are weak positive and negative test results in each batch of test results, such as missed detection.
  • This test requires monitoring and testing during the period of breeding chickens laying eggs. Only the testing of adult chickens can obtain effective testing data and testing results. For young chickens, there is a large detection error. It is worth noting and emphasizing that a variety of cells, components and a variety of immune antibodies in the whole blood of infected breeders can react non-specifically with whole bacterial antigens (non-pure antigens, complex bacterial surface components), and carry non-monoclonal antigens.
  • the antigen of the factor component may also cross-react with a variety of bacterial infection antibodies. In practical applications, it affects the purification effect of the disease and the progress of the purification work. It is a key factor restricting the monitoring and detection of Salmonella pullorum and further eradication and purification!
  • Lipopolysaccharides are extracted from pullorum and Salmonella gallisepticum as antigens.
  • An enzyme-linked immunosorbent assay (ELISA) has also been developed to detect pullorum and Salmonella gallisepticum. This technology can be used to scale serum samples in the laboratory. Infective antibody detection, to a certain extent, overcomes the low sensitivity of the classic plate agglutination reaction, including the low detection rate of antibodies in subclinical infections and chickens with relatively weak immune response capabilities. It has also been reported that purified flagellin, outer membrane protein, and fimbriae protein are used as ELISA detection antigens to detect Salmonella enteritidis infection in chickens.
  • ELISA technology has a certain degree of positive reaction to other Salmonella. It may react positively with Enterobacteriaceae, especially Escherichia coli. At the same time, the purity of flagellin, outer membrane protein, and fimbrin is required to be high, so as to reduce the false positive rate.
  • the classic rapid diagnostic technique for veterinary clinical monitoring, detection and purification and eradication of pullorum and Salmonella typhi infection is the plate agglutination test.
  • the specificity, sensitivity and accuracy of detection results urgently need to be effectively improved and perfected. Therefore, if a special protein in Pullorum and Salmonella gallisepticum can be used, it does not need to be purified and prepared, and this special protein can be selectively used to express and carry on the surface of inert carrier bacteria to directly specifically target Pullorum and Chicken.
  • Antibodies for Salmonella typhi infection ensure the specificity, sensitivity and accuracy of the agglutination test, which will have great application prospects.
  • the inert carrier Salmonella S9 there is no self-coagulation phenomenon, the number of bacteria at the working concentration and the whole blood of different breeders with multiple genetic backgrounds do not undergo non-specific agglutination, and the surface of the bacteria can express and carry a single Antigen factor P, the antigen factor P exists and is expressed in Pullorum and/or Salmonella gallisepticum, and the antigenic factor P specifically targets Pullorum and/or Salmonella gallbladder infection with whole blood antibodies.
  • the inert carrier indirect agglutination test S9-P based on the surface expression of this bacterium and carrying a single antigenic factor P can provide a new type of inert carrier indirect agglutination test pullorum and/or Salmonella typhi.
  • the detection system monitors and detects pullorum and/or Salmonella typhi infection with whole blood antibodies.
  • the detection system is simple to operate, specific, sensitive, fast, and the results are clear and easy to judge.
  • the present invention provides an inert carrier indirect agglutination test detection system.
  • the detection system includes an inert carrier bacteria S9 and a complex S9-P that expresses and carries P antigen factors on its surface.
  • the detection system is an inert carrier strain S9 containing pullorum and/or Salmonella typhi p gene.
  • the detection system is to identify the correct strain by connecting the p gene of Salmonella pullorum to the pBR322 plasmid and then introducing it into the S9 electrotransformation competent cell.
  • the content of the present invention also includes the construction method of the inert carrier indirect agglutination test detection system, including the following steps:
  • the recombinant plasmid p-pBR322 is transformed into S9 electro-transformed competent cells and the recombinant strain is identified as the inert vector indirect agglutination test detection system S9-P.
  • nucleotide sequence of the P antigen factor encoding gene p of Salmonella pullorum in the step 1) is designed by the whole genome sequence design of pullorum pullorum and Salmonella gallisepticum published by NCBI.
  • the gene p is obtained using the Salmonella pullorum CVCC526 reference strain as a template, and the target gene p is obtained by PCR amplification by designing a specific primer pair.
  • the specific primer pair is:
  • the content of the present invention also includes the application of the inert carrier indirect agglutination test detection system in the preparation of reagents or kits for detecting chicken-related pathogen infections.
  • the content of the present invention also includes the application of the inert carrier indirect agglutination test detection system in the preparation of reagents or kits for detecting Salmonella pullorum or Salmonella typhi infection.
  • the content of the present invention also includes a detection kit for pullorum and/or Salmonella typhi, which includes the inert carrier indirect agglutination test pullororia and Salmonella typhoid detection system.
  • the detection kit also includes a positive control and a negative control.
  • the positive control may be a positive serum prepared by artificially infecting SPF with a standard strain of Salmonella pullorum; the negative control is SPF chicken serum.
  • the detection system S9-P of the present invention detects pullorum and/or Salmonella typhi infection serum/whole blood antibodies in chicken flocks, and the inert carrier indirect agglutination test detection system S9-P is based on the surface expression and carrying of inert carrier bacteria S9
  • a single antigenic factor P the antigenic factor P exists and is expressed in pullorum and Salmonella gallisepticum, and its antigenic factor P specifically targets pullorum and Salmonella gallisepticum infection with whole blood antibodies.
  • the inert carrier bacterium S9 is deposited in the China Common Microorganism Collection Management Center (CGMCC), the preservation address is Beijing, China, the preservation number is CGMCC No.17340, the preservation date is March 18, 2019, and the classification is named Salmonella ( Salmonella sp.), strain code S9.
  • CGMCC China Common Microorganism Collection Management Center
  • the inert carrier Salmonella is isolated and screened from healthy chickens.
  • the inert carrier Salmonella bacteria liquid does not self-coagulate.
  • the number of bacteria at the working concentration (the bacterial concentration reaches the order of 10 10 cfu bacteria) is comparable to that of chickens with different genetic backgrounds.
  • Source serum/whole blood does not have non-specific agglutination reaction.
  • Chicken-source serum/whole blood includes but not limited to SPF chicken serum/whole blood, healthy chicken serum/whole blood, positive serum/whole blood of multiple bacterial infection chickens, multiple parasites
  • the results of the agglutination reaction were negative for the positive serum/whole blood of worm-infected chickens, the positive serum/whole blood of multiple virus-infected chickens, and the immune serum/whole blood of various breeds of chickens.
  • the invention relates to a novel inert carrier indirect agglutination test detection system S9-P for preparation, which is a novel inert carrier indirect agglutination test detection system S9-P constructed based on inert carrier bacteria S9 using molecular biology methods.
  • the culture method is as follows: pick a single colony from the preserved strains and streak it in the ampicillin-resistant (100 ⁇ g/mL) LB agar medium.
  • the culture temperature is 37°C. Cultivate for 24h to form off-white round medium-sized colonies.
  • the invention relates to a novel inert carrier indirect agglutination test detection system S9-P.
  • the strain S9-P from the inert carrier indirect agglutination test detection system is amplified and cultured with a certain concentration of ampicillin resistant (100 ⁇ g/mL) LB liquid medium and harvested.
  • Inactivate bacteria adjust the concentration of the bacterial solution, and prepare by staining with a specific ratio of 20% volume of blue material (1 part of crystal violet: 4 parts of Swiss blue), and verify the new inert carrier indirect agglutination test detection system S9 by the test glass plate agglutination test -P does not have self-coagulation phenomenon, S9-P and PBS phosphate buffer solution and other electrolyte solutions do not produce self-coagulation phenomenon. It is compatible with a variety of chicken serum/whole blood from different genetic backgrounds that are not infected with pullorum and Salmonella gallisepticum. Non-specific agglutination reaction does not occur.
  • Chicken-derived serum/whole blood for non-pullaria and Salmonella typhi infection includes but not limited to SPF chicken serum/whole blood, healthy chicken serum/whole blood, a variety of non-pullaria, Salmonella typhi bacteria Infected chicken positive serum/whole blood, multiple parasite-infected chicken positive serum/whole blood, multiple viral infection chicken positive serum/whole blood and various breeds of chicken immune serum/whole blood, S9-P and the above-mentioned serum to be tested /After the equal volume of whole blood is mixed thoroughly, no agglutination particles will appear within two minutes.
  • S9-P and the laboratory-prepared samples of Salmonella pullorum infection positive antibody samples also includes the pullorum positive serum/whole blood and the reference strain of Salmonella pullorum provided by third-party laboratories and related reference laboratories
  • Immune serum/whole blood prepared from artificially infected SPF chickens and laboratory-diagnosed positive serum/whole blood for Salmonella pullorum infection collected from three different breeder farms, S9-P and the above serum/whole blood to be tested in equal volumes are thoroughly mixed. After that, blue agglutinated particles with clear background appeared within two minutes, and the agglutination reaction was positive.
  • the present invention relates to a new type of inert carrier indirect agglutination test detection system S9-P.
  • Simultaneous parallel detection with commercial Salmonella pullorum agglutination antigen 1) When comparing with the ELISA kit of the Dutch Biochek company as the hypothetical gold standard reference method, the inert carrier indirect agglutination test detection system S9-P has a detection coincidence rate (0.62) ), which is significantly better than the detection coincidence rate of commercial agglutination antigen (0.05-0.42); 2) When the bacterial isolation in chicken is used as the hypothetical gold standard reference method, the positive detection rate of bacterial isolation and identification is 22.5% (9/40 ), the positive detection rate of commercial agglutinating antigen is 50% (20/40), and the positive detection rate of S9-P is 25% (10/40).
  • the coincidence rate is more consistent (basically the same, greater than or equal to 90%); 3) When Western blot is used to detect P factor-targeted antibodies and the S9-P test result is verified, the S9-P test result and Western blot test P factor The coincidence rate of the targeted antibody results is consistent, which is 100%.
  • the novel inert carrier indirect agglutination test detection system S9-P of the present invention is applied to the spot detection of whole blood antibodies in chicken flocks with pullorum and Salmonella typhi infection through agglutination test.
  • the new inert carrier indirect agglutination test detection system S9-P reagent 5 ⁇ L ⁇ 10 ⁇ L
  • a drop of chicken blood 5 ⁇ L ⁇ 10 ⁇ L
  • the positive agglutination reaction particles are clearly visible to the naked eye on a white light background. The result must be quickly determined within two minutes.
  • the chickens are screened for pullorum and Salmonella typhi infection.
  • the present invention discovers and finds that antigen factor P specifically exists in pullorum and Salmonella gallisepticum, and its coding gene p is expressed on the surface of inert carrier strain S9 and carries antigen factor P to establish an inert carrier specifically targeting a single antigen factor
  • the detection system for pullorum and Salmonella gallisepticum uses a variety of complex whole bacterial antigens that replace the surface components of the bacteria as the indirect agglutination test diagnostic antigens. It can be accurate and maximized while retaining the advantages of intuitive agglutination reaction results and easy operation.
  • the present invention relates to a novel inert carrier indirect agglutination test detection system S9-P.
  • the inert carrier bacteria replaces pullorum and Salmonella gallisepticum whole bacteria agglutination diagnostic antigen , To avoid the non-specific cross-agglutination reaction produced by multi-component whole bacterial antigens.
  • the present invention discovers and verifies the existence and expression of antigen factor P in Pullorum Gallus gallbladder and Salmonella typhi, and constructs a novel inert carrier indirect agglutination test detection system S9-P based on the surface expression of the inert carrier strain S9 and carrying a single antigen factor P.
  • a drop of chicken blood (5 ⁇ L ⁇ 10 ⁇ L) was collected in the chicken coop next to the pen, and only through naked eye observation, rapid specificity and on-site monitoring of pullorum in chicken flocks, Salmonella typhi infection in chickens.
  • the present invention innovatively establishes a new type of inert carrier indirect agglutination test detection system S9-P, which replaces, effectively improves and perfects the existing Salmonella pullorum agglutination antigen diagnostic test that has been used for many years, and overcomes many years (since the 1950s)
  • Traditional and classic agglutination tests are restricted by the technical bottleneck of specificity and sensitivity, and have good potential application value and market prospects.
  • Figure 1 The sequence and alignment of the complete gene (p gene) encoding Salmonella pullorum antigen factor P: CP012347.1, LK931482.1, CP006575.1 and CP022963.1 are different Salmonella pullorum uploaded by NCBI GenBank gene database The p gene sequence of the strain.
  • Figure 2 The PCR amplification and identification electrophoresis diagram of the main subunit coding gene of antigen factor P (pA gene): Lane M is DL5000 Marker, Lane 1 is Salmonella pullorum isolate SP 0-1; Lane 2 is Salmonella pullorum Isolate S.P1-1, lane 3 is Salmonella pullorum isolate S.P2-1, lane 4 is Salmonella pullorum isolate S.P3-1, lane 5 is Salmonella pullorum isolate SP 4-1, lane 6 is Salmonella pullorum isolate S.P5-1, lane 7 is Salmonella pullorum reference strain CVCC526, lane 8 is Salmonella pullorum reference strain CVCC535, lane 9 is E. coli APEC-XM, lane 10 is E. coli DH5 ⁇ .
  • Lane M is DL5000 Marker
  • Lane 1 is Salmonella pullorum isolate SP 0-1
  • Lane 2 is Salmonella pullorum Isolate S.P1-1
  • lane 3 is Salmonella pullorum isolate S.P2-1
  • FIG. 3 PCR amplification and identification electrophoresis diagram of the complete coding gene of antigen factor P (p gene): Lane M is Trans 2K Plus II Marker, lane 1 is the PCR amplification product of p gene p-PCR, template DNA is from chicken Salmonella pullorum reference strain CVCC526.
  • Figure 4 Gene cloning and p recombinant plasmid containing p-p 19T enzyme digestion electrophoresis: wherein M a is a lane Trans 2K Plus II Marker, Lane M b is Trans 2K Plus Marker, lanes 1-3 are recombinant plasmids 19T -p NheI single restriction enzyme digestion, lanes 4 to 6 are recombinant plasmid 19T-p BamHI single restriction enzyme digestion, lanes 8 to 10 are recombinant plasmid 19T-p NheI and BamHI double restriction restriction enzyme digestion.
  • Figure 5 Cloning of the p gene and identification of p-pBR322 restriction enzyme digestion electrophoresis diagram: Lane M is Trans 15K Marker, Lane 1 is p-pBR322 recombinant plasmid, Lane 2 is p-pBR322NheI single enzyme digestion, lane 3 is the pBR322 plasmid without the p gene, lane 4 is the p-PCR result of the recombinant expression bacterial solution containing the p gene recombinant plasmid, and lane 5 is the p-PCR positive control of the p gene recombinant plasmid.
  • Figure 6 The negative staining transmission electron microscope observation image of the carrier strain S9 and the recombinant carrier strain S9-P expressing the p gene of Salmonella pullorum (Philips Tecnai 12 TEM, 46000 ⁇ ).
  • Figure 7 Image of agglutination reaction between inert carrier detection system S9-P and Salmonella pullorum positive serum.
  • Figure 8 The image of the agglutination reaction between the inert carrier detection system S9-P and Salmonella pullorum positive whole blood.
  • Figure 9 Inert carrier detection system S9-P and commercial agglutination antigen detection results and comparison chart. Area 1: S9-P and positive serum agglutination reaction; Area 2: S9-P and negative serum control agglutination reaction; Area 3: Commercial agglutination antigen and positive serum agglutination reaction; Area 4: Commercial agglutination antigen and Negative serum control agglutination reaction; area 5: agglutination reaction between S9-P and whole blood collected on site; area 6: agglutination reaction of commercial agglutination antigen and whole blood collected on site.
  • FIG 10 The Western blot of the positive serum IgG detected by the inert carrier detection system S9-P that recognizes the P antigen factor and the verification of the inert carrier indirect agglutination test detection system S9-P.
  • the positive sera of S9-P test results include the serum of SPF chicken artificially infected with Salmonella pullorum in the control group, the experimental group 1 and the experimental group 2 from clinical breeder sera (the S9-P test result is positive); the negative serum of the S9-P test result includes The control group was SPF chicken serum, and the experimental group 1 and experimental group 2 were from clinical breeder serum (the S9-P test result was negative).
  • Figure 11 Inert vector detection system S-9 detects SPF chickens with different infection time.
  • the reference strains CVCC526 and CVCC535 of Salmonella pullorum involved in the present invention were purchased from the China Veterinary Microbial Culture Collection and Management Center.
  • SP 4-1, SP 5-1, and SP03 come from clinically ill chickens, obtained through conventional isolation methods, identified as Salmonella pullorum and named them. Different isolates have different names and are stored in the applicant’s laboratory.
  • Salmonella typhi SG01 is isolated and preserved in the laboratory of the patent applicant (see National Invention Patent Application No. 201811491200.6), and the avian-derived Escherichia coli APEC-XM and the engineered Escherichia coli DH5 ⁇ are preserved in the laboratory.
  • the goat anti-mouse IgG-HRP and goat anti-chicken lgG-HRP involved in the present invention were purchased from Soleibao Technology Co., Ltd.; 6 ⁇ Loading Buffer, pfu high-fidelity enzyme, and Trans 2K Plus II Marker were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; rTaq DNA polymerase, restriction endonuclease NheI, and restriction endonuclease BamHI were purchased from TakaraBio; T4 ligase, PMD19-T simple vector were purchased from Promega, USA; 2 ⁇ Taq Master Mix (Dye Plus) Purchased from Nanjing Nuoweizan Biotechnology Co., Ltd.; DNA gel recovery kit was purchased from Tiangen Biochemical Technology Co., Ltd.
  • the Salmonella S9 used in the present invention has been deposited in the China Common Microorganism Collection Management Center (CGMCC), the deposit address is Beijing, China, the deposit number is CGMCC No.17340, the preservation date is March 18, 2019, and the classification is named Salmonella (Salmonella sp.), the strain code is S9.
  • CGMCC Common Microorganism Collection Management Center
  • Salmonella pullorum ATCC 9120 strain (NCBI accession number: CP012347.1), Salmonella pullorum S44987_1 strain (NCBI accession number: LK931482.1), Salmonella pullorum S06004 strain (NCBI accession number: CP006575.1), The full-length genome sequence published by Salmonella pullorum QJ-2D-Sal strain (NCBI accession number: CP022963.1), PCR amplification primers were designed using oligo7 software, and the genomic DNA of Salmonella pullorum was extracted with a nucleic acid extraction kit, including the reference strain CVCC526 , CVCC535, isolates SP 0-1, S.P1-1, S.P2-1, S.P3-1, SP 4-1, SP 5-1, and also added avian Escherichia coli APEC-XM and Escherichia coli engineering Bacteria DH5 ⁇ is a negative control.
  • the PCR amplification results based on the p gene showed that the expected target fragments could be amplified in the Salmonella pullorum strains, but the target fragments could not be amplified in the negative control ( Figure 2).
  • the DNA sequencing results and comparison analysis of the results of Nanjing Kinco Biotechnology Co., Ltd. showed that the p gene sequence is consistent with the report, and there is no mutation base, indicating that The gene is highly conserved in Salmonella pullorum strains.
  • Salmonella pullorum reference strains CVCC526 and CVCC535, and Salmonella pullorum isolates SP 0-1, S.P1-1, S.P2-1, S.P3-1, SP 4-1, SP 5- 1 These isolates come from breeders with pullorum infection, a common type of infectious disease in clinical practice. They are isolated from chickens by conventional isolation methods and identified as Salmonella pullorum. Different isolates have different names, which are different from pullorum. The characteristics of the Salmonella reference strain are the same), the culture broths of avian E. coli APEC-XM and E. coli engineered strain DH5 ⁇ are concentrated by 10 times and then subjected to SDS-PAGE electrophoresis.
  • the protein is transferred to the methanol-activated PVDF membrane and added in sequence
  • Mouse anti-P antigen factor polyclonal antibody IgG is the primary antibody
  • 1:5000 diluted goat anti-mouse IgG-HRP is the secondary antibody
  • Western blot results show that the purpose of P antigen factor
  • the bands are expressed in Salmonella pullorum Salmonella pullorum reference strains CVCC526, CVCC535 and isolates SP 0-1, S.P1-1, S.P2-1, S.P3-1, SP 4-1, SP 5-1
  • E. coli APEC-XM and DH5 ⁇ did not show the target band, indicating that the P antigen factor only exists in Salmonella pullorum.
  • the 10 ⁇ L connection system is as follows: 19T vector 1 ⁇ L, PCR recovery product 4 ⁇ L, solution I solution 5 ⁇ L, and the above reaction system is placed in a 16°C metal bath Connect overnight.
  • the next day chemically transform the ligation product into DH5 ⁇ competent cells.
  • the operation is as follows: place the DH5 ⁇ competent cells on ice to thaw, and add 10 ⁇ L of the ligation product to the competent cells (add the ligation product when the competent cells are just thawed ), flick and mix well, ice bath for 30min; heat shock at 42°C for 30s, immediately place on ice for 2min.
  • P-PCR identification observe the growth of bacteria on the ampicillin LB solid medium overnight, pick a single colony and shake it in ampicillin liquid LB (containing 100 ⁇ g/mL ampicillin), take 2 ⁇ L as a template for PCR identification of bacterial liquid, system: 2 ⁇ rTaq DNA polymerase mix 10 ⁇ L, upstream primer (p-UP) 1 ⁇ L, downstream primer (p-LO) 1 ⁇ L, template (bacterial solution) 2 ⁇ L, deionized water 6 ⁇ L.
  • the reaction conditions were as follows: 95°C for 10 min; 94°C for 1 min, 52°C for 1 min, 72°C for 1 min, 25 cycles; 72°C for 10 min. 1% agarose gel 90V electrophoresis identification.
  • Plasmid digestion identification use the kit to extract the plasmid, single digestion of the plasmid NheI, double digestion with NheI and BamHI, and then electrophoresis identification.
  • NheI single enzyme digestion system M buffer 5 ⁇ L, NheI 1 ⁇ L, plasmid 30 ⁇ L, deionized water 14 ⁇ L.
  • Double enzyme digestion system BglI buffer 5 ⁇ L, NheI 1 ⁇ L, BamHI 1 ⁇ L plasmid 30 ⁇ L, deionized water 13 ⁇ L. It was identified by 90V electrophoresis on 1% agarose gel after 3h in 37°C water bath ( Figure 4).
  • the recombinant bacteria with the correct size of the plasmid was digested with the kit to extract the plasmid, the pBR322 plasmid and 19T-p were double digested, and electrophoresis identified the 4361bp and 4845bp bands respectively.
  • the kit to recover we designed the following T4 connection system: 10 ⁇ buffer solution 1 ⁇ , pBR322 recovery product 2 ⁇ L, p recovery product 2 ⁇ L, T4 ligase 1 ⁇ L, deionized water 4 ⁇ L. Metal bath at 16°C overnight.
  • the p-pBR322 plasmid ligated overnight in the previous step is transformed into S9 electrotransformation competent cells, the specific operation is as follows:
  • Electrotransformation operation Take 2 ⁇ L p-pBR322 plasmid and 40 ⁇ L S9 electrotransformation competent cells and mix, add the above mixture to 0.1cm BioRad electrode cup and shock, after electrotransformation, quickly suck the product into 1mL SOC medium, shake at 37°C for 4h Discard the supernatant at 4000 rpm for 10 minutes, leave a little liquid at the bottom, resuspend the coated ampicillin plate, and incubate overnight at 37°C.
  • strain S9 and recombinant strain S9-P were respectively inoculated on LB and ampicillin-resistant LB agar medium, incubated at 37°C for 24h, and then single colonies were picked and inoculated into LB and ampicillin-resistant LB liquid medium, respectively. After two generations, a small amount of bacterial solution was inoculated into LB and ampicillin-resistant LB liquid medium.
  • Example 2 The constructed strain S9-P expressing factor P was selected from the preservation strains and streaked an appropriate amount on the ampicillin-resistant (100 ⁇ g/mL) LB agar plate medium.
  • the culture temperature was 37°C, and it could form an off-white circle in 24 hours.
  • Medium size colonies. Pick a single colony and inoculate it in ampicillin LB liquid medium at 37°C and shake culture for 16h ⁇ 18h. Transfer the above seed bacteria liquid to 300mL ampicillin LB liquid medium at a ratio of 1:100, then shake and expand at 37°C for 16h ⁇ 18h.
  • the inert carrier indirect agglutination test system S9-P was mixed with a vortexer, and the agglutination test was performed with sterile PBS, physiological saline and SPF chicken serum to ensure that the reagents did not self-agglutinate and did not appear non-specific agglutination.
  • sterile PBS, physiological saline and SPF chicken serum to ensure that the reagents did not self-agglutinate and did not appear non-specific agglutination.
  • test results show that the inert carrier indirect agglutination test detection system S9-P and PBS, physiological saline electrolyte solution does not self-coagulate, and it is compatible with a variety of non-Salmonella pullorum chicken-derived serums of different backgrounds: including SPF chicken serum, non-Salmonella pullorum Bacterial infection chicken positive serum, chicken parasitic infection positive serum, chicken viral infection positive serum, the test results are negative for agglutination, and the results of the positive serum and immune sera from different sources of Salmonella pullorum infection are agglutination. The reaction was positive (Table 2).
  • test results show that the inert carrier indirect agglutination test detection system S9-P and PBS, physiological saline electrolyte solution does not self-coagulate, and it is different from a variety of non-Salmonella pullorum infections with chicken-derived whole blood, SPF chicken whole blood, and non-chicken whole blood.
  • Salmonella pullorum bacterial infection chicken positive whole blood, chicken parasitic infection positive whole blood, chicken viral infection positive whole blood the test results are negative for agglutination, and it reacts with different sources of Salmonella pullorum infection positive serum and immune sera The results were all positive for agglutination (Table 3).
  • the ELISA test kit for Salmonella group D from Biochek in the Netherlands was assumed to be the "gold standard reference", and the domestic commercial agglutination antigen and inert carrier indirect agglutination test detection system S9-P were used for parallel detection of 232 chicken clinically collected sera, respectively using Kappa
  • the test and ROC curve compare the consistency and difference of the three sets of results obtained in the above three methods.
  • the inspection results show that the inert carrier indirect agglutination test detection system S9-P in the agglutination reaction test is fast, the results are clear, easy to judge, and sensitive. It has high performance and specificity, stable and reliable, and the coincidence rate with the results of the hypothetical gold standard reference method is better, and the AUC value is higher and close (Table 4).
  • a SEN sensitivity
  • b SPE specificity
  • c TCR overall coincidence rate
  • d PPV positive predictive value
  • e NPV negative predictive value
  • AUC Vaule the size of the area under the ROC curve.
  • Procedure 1 Add 100 ⁇ L of the buffered PBS liquid of each organ tissue and the immersed organ tissue to 3mL SC (Selenite Cysine Enrichment Solution), and apply 100 ⁇ L of cultured SC bacteria solution to XLD (wood Sugar lysine deoxycholate agar) solid medium.
  • SC Senite Cysine Enrichment Solution
  • the carrying status of Salmonella pullorum in 40 breeder chickens at 70 days of age was systematically separated and identified. If each of the 40 chickens corresponds to each organ sample, if Salmonella pullorum can be isolated, It was determined that the chicken was positive for Salmonella pullorum infection. The test results showed that Salmonella was isolated from 9 out of 40 chickens, and the serotype was Salmonella pullorum. In the experiment, the commercial pullorum/typhoid staining agglutination antigen and inert carrier detection system were used to detect and compare the serum of each chicken respectively.
  • the comparison results showed that: 1In terms of detection rate, Salmonella pullorum bacteria was isolated The detection rate of the identification results is 22.5%, the detection rate of commercial agglutinating antigen is 50%, the detection rate of the inert carrier detection system S9-P is 25%, and the detection rate of the inert carrier detection system S9-P is separated from bacteria. Results The detection coincidence rate is better, the coincidence rate is greater than or equal to 90%; 2In the bacteria isolation negative sample, the inert carrier detection system S9-P detected 4 positive samples. Eliminate the use of antibiotics to cause separation and missed detection; 3If bacteria are isolated from positive samples, 3 copies of the inert carrier detection system S9-P are negative. The analysis considers that a small number of 70-day-old chickens have low antibody titers at this age and lead to detection leakage. It does not rule out that during the window period of bacterial infection, there is no antibody that exceeds the detection limit.
  • Example 6 Inert carrier indirect agglutination test detection system S9-P site chicken whole blood detection and comparative analysis
  • An inert carrier detection system S9-P was used to randomly detect 148 whole blood samples from breeder A flocks on site. 5 ⁇ L-10 ⁇ L whole blood was collected from the chickens to be tested on site and mixed with 5 ⁇ L-10 ⁇ L of different agglutinating diagnostic antigens, and the result was determined within 2min. The results of S9-P were 89 positive (60.14%) and 59 negative (39.86%). The test results showed that the positive rate of Salmonella pullorum infection in the A flock was high.
  • the positive sera tested by S9-P include the control group, which is artificially infected with Salmonella pullorum and SPF chicken serum, the experimental group 1 and the experimental group 2 are from clinical breeder sera (S9-P test is positive); S9-P test negative serum, Including the control group is SPF chicken serum, the experimental group 1 and the experimental group 2 are from clinical breeder serum (S9-P test is negative).
  • Example 8 Inert carrier indirect agglutination test detection system S9-P strain sensitivity test
  • Salmonella typhi SG01 was used to infect SPF chickens at 5 and 19 days of age, Salmonella pullorum SP03 was used to infect 34-day-old SPF chickens, and the inert detection system S9-P strain was used to detect SPF chickens at different infection times.
  • the present invention is a novel inert carrier indirect agglutination test detection system S9-P. Its innovation and advantages are embodied in:
  • the existing commercial agglutination antigen of Salmonella pullorum uses a standard strain of pullorum (or/ (And variant strains) inactivated whole bacteria to prepare agglutinating antigen, theoretically and practically, it has been proved that this kind of agglutinating antigen has cross non-specific agglutination reaction, and the bottleneck restriction of low sensitivity, poor stability and poor reproducibility.
  • the development of the new inert carrier detection system S9-P involved in the present invention is based on the preliminary screening and verification of the inert carrier (bacteria) S9 that has no cross-agglutination reaction with chicken serum from various backgrounds, avoiding non-specific cross-agglutination caused by multi-component whole bacterial antigens Reaction interference.
  • the present invention also discovers and verifies that the antigen factor P is only expressed in Salmonella pullorum species and is highly conserved. It is the first to use an inert carrier (bacteria) based on S9 bacterial surface expression and carry a single antigen factor P to construct a new inert carrier detection system S9-P specifically targets the whole blood or serum of Salmonella pullorum infection.
  • the present invention innovatively improves and maximizes the technical bottleneck of the traditional and classic agglutination tests that have been used for many years (since the 1950s) for the detection of agglutinated antigens and antibodies (instability and poor reproducibility) It has good application value and market prospects.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Urology & Nephrology (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Virology (AREA)
  • Plant Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

提供了惰性载体间接凝集试验检测系统及其应用。具体而言，间接凝集试验检测系统包括惰性载体菌S9和在其表面展呈表达和携带P抗原因子的复合体S9-P。该惰性载体间接凝集试验检测系统仅携带P抗原因子，与鸡白痢、鸡伤寒沙门氏菌感染鸡的全血或血清在一定浓度条件下产生肉眼可见的阳性颗粒凝集反应，而与非鸡白痢、鸡伤寒沙门氏菌感染的不同背景鸡源血清、全血不出现非特异性交叉凝集反应。S9-P基于玻板凝集反应操作平台，凝集反应颗粒肉眼可见，结果清晰易判，在两分钟内完成测试和结果判定。S9-P适用于鸡群中鸡白痢、鸡伤寒沙门氏菌感染的靶向特异性检测，在鸡群的现场监测和诊断中具有很好的应用前景。

Description

惰性载体间接凝集试验检测系统及其应用 技术领域
本发明属于生物医学和免疫诊断检测技术领域，具体涉及惰性载体间接凝集试验检测系统。该间接凝集试验检测系统包括惰性载体沙门氏菌S9表达和携带单一抗原因子P建立的新型惰性载体间接凝集试验鸡白痢和/或鸡伤寒沙门氏菌检测系统S9-P。
背景技术
鸡白痢是由鸡白痢沙门氏菌(Salmonella pullorum)感染引起鸡和火鸡等禽类的细菌性传染病，主要危害三周龄以内雏鸡，死亡率高，成年鸡感染后无明显症状，但可通过种蛋源垂直传播于后代雏鸡，是危害我国养鸡业的重要疾病，鸡伤寒沙门氏菌(Salmonella gallinarum)感染引起鸡和火鸡等禽类的细菌性传染病，也是危害我国养鸡业的重要疾病，我国已将鸡白痢、鸡伤寒沙门氏菌病列为二类动物疫病，鸡白痢也是世界动物卫生组织(OIE)规定为必须通报的动物疫病之一。鉴于这两种病原菌病原特性具有种源垂直传播的特征，集约化养鸡场一旦发生上述疫病会造成巨大的经济损失。鸡群一旦感染，药物难以清除和根治，而病原分离鉴定费时繁琐和耐药菌普遍存在，在临床工作中防治难度很大，因而对该疾病防控最根本的策略只有通过种鸡群的有效监测和根除净化，目前我国已将种鸡场沙门氏菌净化工作列入《国家中长期动物疫病防治规划(2012-2020年)》中。
事实上，鸡白痢、鸡伤寒感染检测方法众多，值得注意的是，基于血清学诊断方法中的平板凝集反应为经典的检测方法，由于其圈栏边鸡舍内鸡全血样品检测的便捷和实用性，同时考虑其廉价、时效性和现场规模化筛选检测适用性，具有无可比拟的诊断应用优势。其原理是细菌颗粒性诊断抗原结合相应的血清抗体后，当有电解质存在、温度适宜时，抗原颗粒和相应特异性抗体结合，出现相互凝集凝聚现象，形成凝集小块或颗粒，仅需通过肉眼就能现场观察和判定结果。我们将参与反应的抗原称为凝集原，抗体称为凝集素。操作时将凝集诊断抗原与含有相应特异抗体的血清或全血各滴一滴于洁净的透明玻璃平板上，等体积轻轻混合后，室温下2分钟内观察和判定反应结果，如出现肉眼可见的颗粒状凝集现象，即为阳性凝集反应。凝集试验适用于病原感染的快速诊断，具有众多优点，如：简便快捷、不需要任何额外(实验室)仪器、成本低廉且可在圈栏边鸡舍内仅需一滴鸡血现场测试等。尽管该凝集抗原检测已经有多年的实践应用，但是在实际应用中也逐渐暴露出存在的局限性和技术瓶颈制约，如目前国内应用最广泛的商品化鸡白痢、鸡伤寒凝集抗原的检测诊断中，被报道存在多种非特异性交叉反应，由于全菌抗原的交叉非特异性因素导致假阳性率高；基于菌体O抗原固有的O不凝集性因素影响，各批次检测结果稳定性不佳和重复性不理想；此外，O抗原寡糖抗原性相对较弱，检测鸡白痢、鸡伤寒沙门氏菌感染鸡全血、血清凝集反应的敏感性瓶颈制约而导致漏检，也注意到该方法仅对成年鸡群(种鸡开产前16周龄)检测相对敏感，对雏鸡则可能存在较大的检测误差。前期研究数据中，专利申请发明人所在实验室使用传统的商品化凝集抗原，分两次在不同时间检测同一批来自某种鸡场200份血清时发现，两批的检测结果总符合率仅为81％，提示每批次检测结果并不稳定，前后一致性不佳。当与荷兰Bio Chek公司的沙门氏菌D群ELISA试剂盒对比检测结果时发现，检测结果总符合率仅为79.5％， 阳性符合率(检出率或特异性)为75.2-79.4％，阴性符合率(敏感性)为79.5-85.5％。上述检测结果和比较分析表明，该商品化凝集抗原检测鸡白痢、鸡伤寒沙门氏菌感染血清、全血抗体时，敏感性、特异性、重复稳定性和结果准确性均有提升的空间和必要性，临床上迫切需要改进现有的凝集试验检测方法，旨在达到较为理想的检测水平。
凝集抗原检测结果准确度有待提高的根本原因和关键环节在于：至今，临床净化工作通常会采用多年来(自上世纪50、60年代开始)一直使用的经典型全血平板凝集试验检测鸡白痢、鸡伤寒沙门氏菌感染(血清或全血抗体)，其检测局限在于凝集抗原由鸡白痢沙门氏菌参考株和(或)变异株组成，为不同菌株混合的全菌抗原。理论上说，这种存在于全菌中多成分的抗原的复合组分，很多成分可能会与肠杆菌同科、属、种，其他科属种细菌和其它成分包括异嗜抗原存在非特异性交叉反应，鉴于凝集抗原制备中需要较高的工作浓度和细菌数量(鸡白痢沙门氏菌凝集抗原浓度已达到10 10cfu细菌数量级)，高抗原浓度引起的非特异性交叉反应弊端必然会影响甚至较为显著干扰监测、检测和诊断结果的准确性。该混合菌的全菌抗原成分复杂，菌体表面暴露抗原成分多样、不单一，许多细菌与鸡白痢、鸡伤寒沙门氏菌具有共同抗原或抗原性极为密切，包括大肠杆菌、微球菌和链球菌(尤其是革兰氏D群链球菌)可感染禽类，当感染种鸡后，与感染鸡群全血中存在的多种成分，理论上存在一定程度的交叉反应，导致了实际检测应用中出现的假阳性结果，事实上，还包括表皮葡萄球菌、产气杆菌、变形杆菌及某些种的亚利桑拉、普罗威登氏菌和枸橼酸菌等，在鸡群全血中存在的非特异性交叉反应可从少数几只鸡到30％-40％；同时考虑到菌体抗原O 1、O 9、O 12三种成分，其寡糖抗原性弱，其中O 12也存在3种变异株，即鸡白痢沙门氏菌中存在标准型、变异型和中间型，导致诊断抗原菌株与感染血清的特异反应针对和匹配性不强，考虑到菌体表面成分多种复杂，O抗原在菌体表面空间构象影响和展呈受限，导致该方法检测的敏感性不高，各批次检测结果中存在弱阳性和阴性检测结果中出现漏检等情况。该试验需要在种鸡产种蛋期间监测、检测，仅对成年鸡群检测才能获得有效检测数据和测试效果，对雏鸡则存在较大检测误差。值得注意和强调的是，感染种鸡体内全血中多种细胞、成分和多种免疫抗体可以与全菌抗原(非纯净抗原，菌体表面成分多种复杂)发生非特异性反应，携带非单因子成分的抗原也可能与多种、属细菌感染抗体存在交叉反应，实际应用中影响疫病净化效果和净化工作的推进，是制约鸡白痢沙门氏菌监测检测和进一步根除净化的关键因素！
据上所述和分析，可以共识到鸡白痢沙门氏菌感染种鸡根除净化的有效启动、进程进度和完成质量都依赖于净化关键技术。目前，面对该重要种源性疾病监测、检测和净化亟需一套简便、快速、廉价并且现场检测敏感、特异的检测技术和净化程序，鉴于种鸡场鸡白痢沙门氏菌净化根除工作顺利开展迫在眉睫，净化检测关键技术的建立和应用对养禽业的长足发展，禽蛋产品质量的安全保障，食品公共卫生和人类健康都十分必要。
从鸡白痢、鸡伤寒沙门氏菌中提取脂多糖作为抗原，也已发展了用于检测鸡白痢、鸡伤寒沙门氏菌的酶联免疫吸附试验(ELISA)，该项技术可用于在实验室规模化血清样本的感染抗体检测，在一定程度上克服上述经典平板凝集反应敏感性不高,包括在亚临床感染和免疫应答能力相对较弱的雏鸡感染体内抗体检出率不高。也有报道采用纯化的鞭毛蛋白、外膜蛋白、菌毛蛋白作为ELISA检测抗原，用于检测肠炎沙门氏菌感染鸡群，但与其他血清学试验一样，ELISA技术 对其他沙门氏菌呈一定程度的阳性反应，也与肠杆菌科细菌尤其包括大肠杆菌可能呈阳性反应。同时鞭毛蛋白、外膜蛋白、菌毛蛋白的纯度要求高，这样才能减少出现的假阳性率。
目前兽医临床上监测、检测并用于净化、根除鸡白痢、鸡伤寒沙门氏菌感染的经典型快速诊断技术即平板凝集试验的特异性、敏感性和检测结果准确性迫切需要有效提高和完善。因此，如果能利用鸡白痢、鸡伤寒沙门氏菌中存在的一种特有蛋白，不需要纯化制备，且能通过选择使用这种特有蛋白在惰性载体菌表面表达和携带，直接特异靶向鸡白痢、鸡伤寒沙门氏菌感染抗体，则确保凝集试验的特异性、敏感性和检测结果准确性，将具备巨大的应用前景。
发明内容
发明目的：针对惰性载体沙门氏菌S9不存在自凝现象，在工作浓度下的细菌数量与多种遗传背景的不同种鸡源全血不发生非特异性凝集反应，且该菌体表面能表达和携带单一抗原因子P，该抗原因子P存在并在鸡白痢和/或鸡伤寒沙门氏菌中表达，且抗原因子P特异靶向鸡白痢和/或鸡伤寒沙门氏菌感染全血抗体。基于该菌表面表达和携带单一抗原因子P建立的惰性载体间接凝集试验鸡白痢和/或鸡伤寒沙门氏菌检测系统S9-P，能提供一种新型惰性载体间接凝集试验鸡白痢和/或鸡伤寒沙门氏菌检测系统监测检测鸡白痢和/或鸡伤寒沙门氏菌感染全血抗体，该惰性载体间接凝集试验鸡白痢和/或鸡伤寒沙门氏菌检测系统操作简便，反应特异、敏感、快速，结果清晰易判，实施在圈栏旁鸡舍内采集一滴鸡血(5μL～10μL)，通过肉眼观察，现场2分钟内快速判定凝集反应结果，监测、检测鸡群中鸡白痢和/或鸡伤寒沙门氏菌感染，特异、敏感。
技术方案：为了解决上述技术问题，本发明提供了惰性载体间接凝集试验检测系统，所述检测系统包括惰性载体菌S9和在其表面展呈表达和携带P抗原因子的复合体S9-P。
其中，所述检测系统是含有鸡白痢和/或鸡伤寒沙门氏菌p基因的惰性载体菌S9。
其中，所述检测系统是通过将鸡白痢沙门氏菌p基因与pBR322质粒连接后导入S9电转化感受态细胞中鉴定正确的菌株。
本发明内容还包括所述的惰性载体间接凝集试验检测系统的构建方法，包括以下步骤：
1)鸡白痢沙门氏菌中P抗原因子编码基因p的获得；
2)鸡白痢沙门氏菌中P抗原因子编码基因p与pBR322质粒的连接获得重组质粒；
3)重组质粒p-pBR322转化入S9电转化感受态细胞鉴定得到重组菌株即为惰性载体间接凝集试验检测系统S9-P。
其中，所述步骤1)中的鸡白痢沙门氏菌中P抗原因子编码基因p的核苷酸序列是由NCBI公布的鸡白痢、鸡伤寒沙门氏菌全基因组序列设计。
其中，所述步骤1)中基因p的获得是以鸡白痢沙门氏菌CVCC526参考株为模板，通过设计特异性引物对进行PCR扩增得到目的基因p。
其中，所述特异性引物对为：
p-UP：5'-ATG AAA CGT TCA CTT ATT GCT GCT-3'
p-LO:5'-TTAA TCA GTT AAT ACC GTC ATC GTC AG-3'。
本发明内容还包括所述的惰性载体间接凝集试验检测系统在制备鸡相关病原感染检测用试剂或试剂盒中的应用。
本发明内容还包括所述的惰性载体间接凝集试验检测系统在制备鸡白痢沙门氏菌或鸡伤寒沙门氏菌感染检测用试剂或试剂盒中的应用。
本发明内容还包括一种鸡白痢和/或鸡伤寒沙门氏菌检测试剂盒，所述检测试剂盒包括所述的惰性载体间接凝集试验鸡白痢、鸡伤寒沙门氏菌检测系统。
其中，所述检测试剂盒还包括阳性对照和阴性对照。
其中，所述阳性对照可以为鸡白痢沙门氏菌标准株人工感染SPF制备得到的阳性血清；阴性对照为SPF鸡血清。
本发明的检测系统S9-P检测鸡群中鸡白痢和/或鸡伤寒沙门氏菌感染血清/全血抗体，所述的惰性载体间接凝集试验检测系统S9-P是基于惰性载体菌S9表面表达和携带单一抗原因子P，该抗原因子P存在并在鸡白痢、鸡伤寒沙门氏菌中表达，其抗原因子P特异靶向鸡白痢、鸡伤寒沙门氏菌感染全血抗体。其中，该惰性载体菌S9保藏于中国普通微生物菌种保藏管理中心(CGMCC)，保藏地址为中国北京，保藏编号为CGMCC No.17340，保藏日期为2019年3月18日，分类命名为沙门氏菌(Salmonella sp.)，菌株代号S9。该菌株保藏证明参见申请号为2019104243698的专利申请。所述的惰性载体沙门氏菌从健康鸡群分离筛选得到，惰性载体沙门氏菌菌液不存在自凝现象，在工作浓度下的细菌数量(细菌浓度达到10 10cfu细菌数量级)与不同遗传背景的多种鸡源血清/全血不发生非特异性凝集反应，鸡源血清/全血包括但不限于SPF鸡血清/全血、健康鸡血清/全血、多种细菌感染鸡阳性血清/全血、多种寄生虫感染鸡阳性血清/全血、多种病毒性感染鸡阳性血清/全血和各种品种鸡免疫血清/全血，其凝集反应结果都为阴性。
本发明涉及的制备用新型惰性载体间接凝集试验检测系统S9-P，是利用分子生物学方法基于惰性载体菌S9构建的新型惰性载体间接凝集试验检测系统S9-P，能在含氨苄青霉素(100μg/mL)抗生素的LB琼脂培养基中培养生长，培养方法如下：从保存的菌种中挑取单个菌落划线于氨苄抗性(100μg/mL)LB琼脂培养基中，培养温度为37℃，培养24h可形成灰白色圆形中等大小菌落。
本发明涉及的新型惰性载体间接凝集试验检测系统S9-P，来自惰性载体间接凝集试验检测系统的菌株S9-P，用氨苄抗性(100μg/mL)LB液体培养基扩增培养一定浓度后收获，灭活细菌，调整菌液浓度，通过20％体积的特定比例混合蓝料(1份结晶紫：4份瑞士蓝)染色制备，通过测试玻板凝集试验验证新型惰性载体间接凝集试验检测系统S9-P不存在自凝现象，S9-P与PBS磷酸盐缓冲液等多种电解质溶液不产生自凝现象，与不同遗传背景的多种非鸡白痢、鸡伤寒沙门氏菌感染的鸡源血清/全血不发生非特异性凝集反应，非鸡白痢、鸡伤寒沙门氏菌感染的鸡源血清/全血包括但不限于SPF鸡血清/全血、健康鸡血清/全血、多种非鸡白痢、鸡伤寒沙门氏菌细菌感染鸡阳性血清/全血，多种寄生虫感染鸡阳性血清/全血，多种病毒性感染鸡阳性血清/全血和各种品种鸡免疫血清/全血，S9-P与上述待检血清/全血等体积充分混匀后，两分钟之内不出现任何凝集反应颗粒。
当S9-P与实验室制备的鸡白痢沙门氏菌感染阳性抗体样本(血清或全血)，也包括来源第三方实验室和相关参考实验室提供的鸡白痢阳性血清/全血、鸡白痢沙门氏菌参考株人工感染SPF鸡制备免疫血清/全血和在三家不同种鸡场采集的经实验室诊断为鸡白痢沙门氏菌感染阳性血清/全血，S9-P与上述待检血清/全血等体积充分混匀后，在两分钟之内出现背景清晰的蓝色凝集颗粒，凝集反应阳性。
本发明涉及提供的新型惰性载体间接凝集试验检测系统S9-P，通过检测圈栏边鸡舍内采集的鸡群血清/全血并比较验证发现：检测一滴鸡全血时，产生的凝集颗粒阳性反应迅速、结果清晰易判。与商品化鸡白痢沙门氏菌凝集抗原作同步平行检测：1)当以荷兰Bio chek公司的ELISA试剂盒作为假定金标准参考方法比较时，惰性载体间接凝集试验检测系统S9-P的检测符合率(0.62)，显著优于商品化凝集抗原的检测符合率(0.05-0.42)；2)当以鸡体内细菌分离作为假定金标准参考方法时，细菌分离鉴定的阳性检出率为22.5％(9/40)，商品化凝集抗原的阳性检出率为50％(20/40)，S9-P的阳性检出率为25％(10/40)，S9-P检测系统与细菌分离鉴定的结果和检出符合率更一致(基本一致,大于等于90％)；3)当以Western blot检测P因子靶向的抗体，对S9-P检测结果进行验证时，S9-P检测结果和Western blot检测P因子靶向抗体结果的符合率一致，为100％。
本发明所述的新型惰性载体间接凝集试验检测系统S9-P，通过凝集试验应用于鸡群中鸡白痢、鸡伤寒沙门氏菌感染全血抗体的现场检测。在玻板凝集试验简易操作平台上，当新型惰性载体间接凝集试验检测系统S9-P试剂(5μL～10μL)与圈栏旁鸡舍内鸡静脉采集的一滴鸡血(5μL～10μL)等体积充分混匀后，在白光背景下肉眼清晰可见阳性凝集反应颗粒，必须在两分钟之内快速判定结果，根据凝集阳性反应监测检测和判定筛选鸡为鸡白痢、鸡伤寒沙门氏菌感染。
有益效果：本发明发现并找到抗原因子P特异性存在鸡白痢、鸡伤寒沙门氏菌中，且其编码基因p在惰性载体菌S9表面表达和携带抗原因子P，建立特异靶向单一抗原因子的惰性载体鸡白痢、鸡伤寒沙门氏菌检测系统，以替代菌体表面成分多种复杂的全菌抗原作为间接凝集试验诊断抗原，在保留凝集反应结果直观，操作简便等众多优势的前提下，能精确和最大程度地完善提高间接凝集抗原反应的特异性和敏感性，替换原来的全血平板凝集反应检测的经典平台系统，提供病原感染(全血抗体)现场快速、特异、敏感监测诊断技术和方法。
本发明涉及提供的新型惰性载体间接凝集试验检测系统S9-P，基于申请人对惰性载体菌S9的发现和潜在研发前景，该惰性载体(菌)替代鸡白痢、鸡伤寒沙门氏菌全菌凝集诊断抗原，避免了多成分全菌抗原产生的非特异性交叉凝集反应。本发明发现并验证了抗原因子P存在并表达于鸡白痢、鸡伤寒沙门氏菌，使用基于惰性载体菌S9表面表达展呈和携带单一抗原因子P而构建新型惰性载体间接凝集试验检测系统S9-P，特异性靶向鸡白痢、鸡伤寒沙门氏菌感染鸡全血抗体，实施在圈栏旁鸡舍内采集一滴鸡血(5μL～10μL)，仅通过肉眼观察、快速特异和现场监测鸡群中鸡白痢、鸡伤寒沙门氏菌感染。本发明创新性建立新型惰性载体间接凝集试验检测系统S9-P，替代、有效改进和完善现有的多年来一直使用的鸡白痢沙门氏菌凝集抗原诊断试验，克服多年来(自上世纪50年代以来)传统、经典凝集试验的特异性和敏感性技术瓶颈制约，具有很好的潜在应用价值和市场前景。
附图说明
图1、编码鸡白痢沙门氏菌抗原因子P的完整基因(p基因)序列和比对示意图：其中CP012347.1、LK931482.1、CP006575.1和CP022963.1为NCBI GenBank基因数据库上传的不同鸡白痢沙门氏菌菌株的p基因序列。
图2：抗原因子P的主要亚单位编码基因(pA基因)的PCR扩增鉴定电泳图：其中泳道M为DL5000 Marker，泳道1为鸡白痢沙门氏菌分离株S.P 0-1；泳道2为鸡白痢沙门氏菌分离株S.P1-1，泳道3为鸡白痢沙门氏菌分离株S.P2-1， 泳道4为鸡白痢沙门氏菌分离株S.P3-1，泳道5为鸡白痢沙门氏菌分离株S.P 4-1，泳道6为鸡白痢沙门氏菌分离株S.P5-1，泳道7为鸡白痢沙门氏菌参考株CVCC526，泳道8为鸡白痢沙门氏菌参考株CVCC535，泳道9为大肠杆菌APEC-XM，泳道10为大肠杆菌DH5α。
图3：抗原因子P的完整编码基因(p基因)的PCR扩增鉴定电泳图：其中泳道M为Trans 2K Plus II Marker，泳道1为p基因的PCR扩增产物p-PCR，模板DNA来自鸡白痢沙门氏菌参考株CVCC526。
图4：p基因的克隆和含p基因重组质粒19T-p酶切鉴定电泳图：其中泳道M a为Trans 2K Plus II Marker，泳道M b为Trans 2K Plus Marker，泳道1～3为重组质粒19T-p NheI单酶切，泳道4～6为重组质粒19T-p BamHI单酶切，泳道8～10为重组质粒19T-p NheI和BamHI双酶切。
图5：p基因的克隆和含p基因重组质粒p-pBR322酶切鉴定电泳图：其中泳道M为Trans 15K Marker，泳道1为p-pBR322重组质粒，泳道2为p-pBR322NheI单酶切，泳道3为不含p基因的pBR322质粒，泳道4为含p基因重组质粒的重组表达菌液p-PCR结果，泳道5为含p基因重组质粒的p-PCR阳性对照。
图6：载体菌S9和表面表达鸡白痢沙门氏菌p基因的重组载体菌S9-P负染透射电镜观察图(Philips Tecnai 12透射电镜TEM，46000×)。
图7：惰性载体检测系统S9-P与鸡白痢沙门氏菌阳性血清凝集反应图象。
图8：惰性载体检测系统S9-P与鸡白痢沙门氏菌阳性全血凝集反应图象。
图9：惰性载体检测系统S9-P与商品化凝集抗原检测结果和对比图。区域1：为S9-P与阳性血清凝集反应；区域2：为S9-P与阴性血清对照凝集反应；区域3：为商品化凝集抗原与阳性血清凝集反应；区域4：为商品化凝集抗原与阴性血清对照凝集反应；区域5：为S9-P与现场采集的全血凝集反应；区域6：为商品化凝集抗原与现场采集的全血凝集反应。
图10：惰性载体检测系统S9-P检测的阳性血清IgG识别P抗原因子的Western blot图和惰性载体间接凝集试验检测系统S9-P验证。S9-P检测结果阳性血清包括对照组为鸡白痢沙门氏菌人工感染SPF鸡血清，实验组1和实验组2来自临床种鸡血清(S9-P检测结果为阳性)；S9-P检测结果阴性血清包括对照组为SPF鸡血清，实验组1和实验组2来自临床种鸡血清(S9-P检测结果为阴性)。
图11：惰性载体检测系统S-9检测不同感染时间的SPF鸡结果。
具体实施方式
本发明中涉及的鸡白痢沙门氏菌参考株CVCC526、CVCC535购自中国兽医微生物菌种保藏管理中心，鸡白痢沙门氏菌分离株S.P 0-1、S.P1-1、S.P2-1、S.P3-1、S.P 4-1、S.P 5-1、SP03来自临床发病鸡群，通过常规分离方法获取，鉴定为鸡白痢沙门氏菌并命名，不同的分离株有不同的名称，在申请人实验室保存，鸡伤寒沙门氏菌SG01为专利申请人实验室分离保存(见国家发明专利申请号201811491200.6)，禽源大肠杆菌APEC-XM和大肠杆菌工程菌DH5α为本实验室保存。
本发明中涉及的羊抗鼠IgG-HRP、羊抗鸡lgG-HRP购自索莱宝科技有限公司；6×Loading Buffer、pfu高保真酶、Trans 2K Plus II Marker购于北京全式金生物技术有限公司；rTaq DNA聚合酶、限制性内切酶NheI、限制性内切酶BamHI购于TakaraBio公司；T4连接酶、PMD19-T simple vector购于美国Promega公司；2×Taq Master Mix(Dye Plus)购于南京诺唯赞生物科技有限公司；DNA凝胶回 收试剂盒购自天根生化科技有限公司。
不同背景多种鸡源血清/全血为本课题组收集保存。
本发明中采用的沙门氏菌S9已经保藏于中国普通微生物菌种保藏管理中心(CGMCC)，保藏地址为中国北京，保藏编号为CGMCC No.17340，保藏日期为2019年3月18日，分类命名为沙门氏菌(Salmonella sp.)，菌株代号S9。
实施例1鸡白痢沙门氏菌P抗原因子的鉴定与验证
通过NCBI上GenBank数据库查找已上传的鸡白痢沙门氏菌中P抗原因子编码基因P(表1)，使用DNAMAN Windows版软件对下载的p基因序列进行比对，结果显示在不同的鸡白痢沙门氏菌菌株中p基因序列高度保守(图1)。事实上，也与我们实验室分离鉴定和经DNA序列测定的多株鸡白痢沙门氏菌分离株p基因序列高度保守。
表1鸡白痢沙门氏菌序列信息
Figure PCTCN2020140030-appb-000001
根据NCBI GenBank中鸡白痢沙门氏菌ATCC 9120株(NCBI登录号：CP012347.1)、鸡白痢沙门氏菌S44987_1株(NCBI登录号：LK931482.1)、鸡白痢沙门氏菌S06004株(NCBI登录号：CP006575.1)、鸡白痢沙门氏菌QJ-2D-Sal株(NCBI登录号：CP022963.1)公布的基因组序列全长，使用oligo7软件设计PCR扩增引物，用核酸提取试剂盒提取鸡白痢沙门氏菌基因组DNA，包括参考株CVCC526、CVCC535，分离株S.P 0-1、S.P1-1、S.P2-1、S.P3-1、S.P 4-1、S.P 5-1，同时增设禽大肠杆菌APEC-XM和大肠杆菌工程菌DH5α为阴性对照。基于p基因的PCR扩增结果显示在鸡白痢沙门氏菌菌株中均能扩增到预期的目的片段，而在阴性对照中未能扩增到目的片段(图2)。使用pfu高保真DNA聚合酶经PCR扩增获得靶向目的片段，南京擎科生物科技有限公司DNA测序结果和结果比对分析表明，p基因序列与报道一致，未有任何突变碱基，说明该基因在鸡白痢沙门氏菌菌种菌株内高度保守。
将鸡白痢沙门氏菌参考株CVCC526、CVCC535，实验室保存的鸡白痢沙门氏菌分离株S.P 0-1、S.P1-1、S.P2-1、S.P3-1、S.P 4-1、S.P 5-1(这些分离株来自鸡白痢感染发病种鸡，在临床上常见一类感染疾病，通过常规的分离方法从鸡体内分离并鉴定为鸡白痢沙门氏菌，不同的分离株有不同的名称，与鸡白痢沙门氏菌参考株的特性一致)，禽大肠杆菌APEC-XM和大肠杆菌工程菌DH5α的培养液经10倍浓缩制样后进行SDS-PAGE电泳，将蛋白转印至甲醇活化的PVDF膜上，依次加入小鼠抗P抗原因子的多抗IgG为一抗，1:5000稀释的羊抗鼠IgG-HRP为二抗，孵育，DAB底物显色，Western blot免疫印迹法结果显示，P抗原因子的目的条带表达在鸡白痢沙门氏菌鸡白痢沙门氏菌参考株CVCC526、CVCC535和分离株S.P 0-1、S.P1-1、S.P2-1、S.P3-1、S.P 4-1、S.P 5-1中，而大肠杆菌APEC-XM和DH5α未显示目的条带，说明P抗原因子仅存在于鸡白痢沙门氏菌中。
实施例2惰性载体间接凝集试验检测系统S9-P的构建
根据NCBI中鸡白痢沙门氏菌p基因全长片段，用Olige7软件设计扩增引 物，分别在上下游引物的5’端加上NheI和BamHI酶切位点及保护性碱基，上下游引物分别为：
p-UP：5'-ATG AAA CGT TCA CTT ATT GCT GCT-3'
p-LO:5'-TTA A TCA GTT AAT ACC GTC ATC GTC AG-3'；
用煮沸法中制备CVCC526鸡白痢沙门氏菌模板，p-PCR体系：5×pfu DNA聚合酶buffer 10μL，dNTP 5μL，上游引物2μL，下游引物2μL，模板2μL，pfu高保真酶(2.5units/uL)2μL，去离子水27μL。PCR反应条件：94℃5min，94℃1min，52℃1min，72℃5min，30个循环，72℃10min。在上述PCR反应结束后，在体系中加rTaqDNA聚合酶(5U/μL)2.4μL，72℃20min加A尾。
PCR扩增产物中加入10μL 6×Loading buffer，用1％琼脂糖凝胶90V电泳1h，紫外凝胶成像仪照胶后切下目的条带，成功扩增出添加了NheI和BamHI两个酶切位点的p基因片段(图3)，说明书操作用琼脂糖凝胶回收试剂盒回收PCR产物，回收产物可于-20℃保存备用。
将上一步获得的加A尾的PCR产物与PMD19-T simple vector载体连接，10μL的连接体系如下：19T载体1μL，PCR回收产物4μL，solution I溶液5μL，将上述反应体系置于16℃金属浴连接过夜。
第二天将连接产物化学转化入DH5α感受态细胞中，操作如下：将DH5α感受态细胞置于冰上解冻，加10μL连接产物于感受态细胞中(在感受态细胞刚刚解冻的时候加入连接产物)，轻弹混匀，冰浴30min；42℃热激30s，立即置于冰上2min。加入250μL平衡至室温的LB，37℃200rpm孵育2h，4000rpm，1min弃上清，留少许上清(约100μL)重悬菌体，涂布于氨苄LB固体培养基(含100μg/mL氨苄青霉素)37℃过夜。
P-PCR鉴定：过夜后观察氨苄LB固体培养基上细菌生长情况，挑取单菌落于氨苄液体LB(含100μg/mL氨苄青霉素)中摇菌，取2μL为模板进行菌液PCR鉴定，体系：2×rTaq DNA聚合酶mix 10μL，上游引物(p-UP)1μL，下游引物(p-LO)1μL，模板(菌液)2μL，去离子水6μL。反应条件如下：95℃10min；94℃1min，52℃1min，72℃1min，25个循环；72℃10min。1％琼脂糖凝胶90V电泳鉴定。
质粒酶切鉴定：使用试剂盒提取质粒，对质粒NheI单酶切，NheI和BamHI双酶切，之后电泳鉴定。NheI单酶切体系：M buffer 5μL，NheI 1μL，质粒30μL，去离子水14μL。双酶切体系：BglI buffer 5μL，NheI 1μL，BamHI 1μL质粒30μL，去离子水13μL。37℃水浴3h后1％琼脂糖凝胶90V电泳鉴定(图4)。
对于上步中p-PCR反应阳性，酶切质粒大小正确的重组菌用试剂盒提取质粒，分别对pBR322质粒和19T-p双酶切，电泳鉴定分别切下4361bp和4845bp处条带的胶块，用试剂盒回收，我们设计如下T4连接体系：10×buffer溶液1μ，pBR322回收产物2μL，p回收产物2μL，T4连接酶1μL，去离子水4μL。16℃金属浴过夜。
将上步连接过夜的p-pBR322质粒转化入S9电转化感受态细胞中，具体操作如下：
电转化感受态细胞的制备：挑取过夜生长的LB平板上的S9单菌落，接种到4mLLB液体培养基中，于37℃振摇3h～5h，观察细菌生长情况。将菌液1:100接种到4mL液体LB培养基中，37℃振摇至OD 600nm到0.4～0.6后冰浴30min，冰浴后4℃4000rpm离心10min，弃去上清。加入预冷的10％甘油洗涤三次，最 后用40μL重悬，可暂存于-20℃备用。
电转化操作：取2μLp-pBR322质粒与40μL S9电转化感受态细胞混合，将上述混合物加入0.1cm的BioRad电极杯中电击，电转后迅速将产物吸到1mLSOC培养基中，37℃振摇恢复4h后4000rpm 10min弃上清，留少许底部液体重悬涂氨苄平板，37℃培养过夜。
第二天观察细菌生长情况，可用p-PCR鉴定和质粒酶切鉴定(图5)，挑取鉴定含P因子正确构建的重组S9-P菌株单菌落，LB培养基扩增后，使用50％甘油与S9-P菌液1:1等体积混合保存于-70℃。
实施例3惰性载体检测系统菌株表达P因子的验证
将菌株S9和重组菌株S9-P单菌落分别接种于LB和氨苄抗性LB琼脂培养基上，置37℃培养24h后挑取单菌落分别接种于LB和氨苄抗性LB液体培养基中，盲传两代后，吸取少量菌液分别接种于LB和氨苄抗性LB液体培养基中，静置培养37℃48h后，10000rpm离心2min，用灭菌PBS重悬沉淀，吸取少量上清液体，悬浮于铜网，并用磷钨酸负染5min，Philips Tecnai 12透射电镜TEM观察拍摄和结果初步显示，S9表面似乎看不出P抗原因子成分，而惰性载体检测系统S9-P菌株表面出现并携带了一种抗原成分——P因子成分(图6)。
实施例4惰性载体间接凝集试验检测系统S9-P的测试和验证
实施例2构建的表达P因子的菌株S9-P保存菌种中挑取适量划线于氨苄抗性(100μg/mL)LB琼脂平板培养基中，培养温度为37℃，24h可形成灰白色圆形中等大小菌落。挑单菌落接种于氨苄LB液体培养基37℃振摇培养16h～18h，上述种子菌液按1:100转接到300mL氨苄LB液体培养基中37℃振摇扩增培养16h～18h后，培养物经4℃离心机8000rpm离心10min，0.4％甲醛生理盐水洗两次后重悬，置于4℃冰箱灭活24h后，再次离心和菌体重悬至0.25％甲醛生理盐水和适宜工作浓度，通过20％体积的特定比例混合蓝料(1份结晶紫：4份瑞士蓝)染色，制成惰性载体间接凝集试验检测系统S9-P，置于4℃保存。
检测前将惰性载体间接凝集试验检测系统S9-P用涡旋仪混匀，先用无菌PBS、生理盐水和SPF鸡血清进行凝集试验确保试剂无自凝和不出现非特异性凝集现象。在超净台内(20℃～25℃)取表面洁净普通玻板若干块，用微量移液器吸取一滴约10μL S9-P垂直滴于水平放置的玻板表面上。随后迅速滴加等量的待检血清。用灭菌枪头将试剂与血清充分混合均匀，涂布成直径1cm～2cm的片状后随即平稳摇动玻板，明确在2min内观察和判定试验结果。标准判定状况为室温下2min内，以S9-P与待检血清产生天蓝色凝集颗粒则凝集反应结果判定为阳性，否则判定为阴性(图7)。
测试结果显示，惰性载体间接凝集试验检测系统S9-P与PBS、生理盐水电解质溶液不出现自凝现象，与不同背景的多种非鸡白痢沙门氏菌鸡源血清：包括SPF鸡血清、非鸡白痢沙门氏菌细菌性感染鸡阳性血清、鸡寄生虫感染阳性血清、鸡病毒性感染阳性血清，其检测的结果都为凝集反应阴性，而与不同来源的鸡白痢沙门氏菌感染阳性血清和免疫血清反应结果都为凝集反应阳性(表2)。
表2惰性载体间接凝集试验检测系统S9-P与鸡源血清反应的测试结果
Figure PCTCN2020140030-appb-000002
Figure PCTCN2020140030-appb-000003
注：“-”表示阴性；“+”表示阳性
当检测全血时，将惰性载体间接凝集试验检测系统S9-P用涡旋仪混匀，先用无菌PBS、生理盐水和SPF鸡血清分别进行凝集试验测试，确保试剂无自凝和不出现非特异性凝集现象。在室温条件下(20℃～25℃)取表面洁净普通玻板若干块，用微量移液器吸取一滴约10μLS9-P垂直滴于水平放置的玻板表面上。随后迅速滴加鸡场现场采集的全血(约10μL)或者抗凝血(约10μL)。用灭菌滴管将试剂与全血充分混合均匀，涂布成直径1cm～2cm的片状后随即平稳摇动玻板，明确在2min内必须观察和判定试验结果。标准判定状况为室温下2min内，以S9-P与待检血清产生蓝色凝集颗粒则反应结果判定为凝集反应阳性，否则判定为凝集反应阴性(图8)。
测试结果显示，惰性载体间接凝集试验检测系统S9-P与PBS、生理盐水电解质溶液不出现自凝现象，与不同背景的多种非鸡白痢沙门氏菌感染鸡源全血、SPF鸡全血、非鸡白痢沙门氏菌细菌性感染鸡阳性全血、鸡寄生虫感染阳性全血、鸡病毒性感染阳性全血，其检测的结果都为凝集反应阴性，与不同来源的鸡白痢沙门氏菌感染阳性血清和免疫血清反应结果都为凝集反应阳性(表3)。
表3惰性载体间接凝集试验检测系统S9-P与鸡源全血测试结果
Figure PCTCN2020140030-appb-000004
注：“-”表示阴性；“+”表示阳性
实施例5惰性载体间接凝集试验检测系统S9-P菌株的临床血清检测和验证
以荷兰Bio chek公司D群沙门氏菌ELISA检测试剂盒假定为“金标准参考”，同时使用国内商品化凝集抗原和惰性载体间接凝集试验检测系统S9-P平行检测232份鸡临床采集血清，分别使用Kappa测验和ROC曲线比较上述三种方法中分别获得三组结果的一致性和差异性，检查结果显示惰性载体间接凝集试验检测系统S9-P在凝集反应测试中，反应迅速、结果清晰易判，敏感性高和特异性稳定可靠，与假定金标准参考方法结果的符合率更好，AUC值更高和接近(表4)。
Figure PCTCN2020140030-appb-000005
Figure PCTCN2020140030-appb-000006
表4：S9-P检测232份临床血清性能的验证和比较
注： aSEN：敏感性； bSPE：特异性； cTCR：总体符合率； dPPV：阳性预测值； eNPV：阴性预测值；AUC Vaule：ROC曲线下方的面积大小。
从某种鸡场随机挑选40只70日龄蛋鸡，分别编号1～40。无菌采集每一只鸡的各个脏器组织于单独的无菌小管中(预先加入无菌PBS，各脏器组织编号见表5)，分别使用两种优化程序进行沙门氏菌分离鉴定，程序一：将各个脏器组织和浸泡脏器组织的缓冲PBS液体100μL加入3mL SC(亚硒酸盐胱氨酸增菌液)增菌液中，过夜培养后取100μL培养SC菌液涂布于XLD(木糖赖氨酸脱氧胆盐琼脂)固体培养基。鉴于沙门氏菌在XLD上的菌落形态为粉红色，带或不带黑色中心(H 2S产生)，而大肠杆菌等可发酵乳糖和木糖的革兰氏阴性菌则因大量产酸而使pH值降低，使指示剂苯酚红变黄，我们首先挑取XLD上疑似粉红色单菌落，在麦康凯平板上划线培养。由于沙门氏菌不发酵乳糖，可与发酵乳糖产酸使中性红指示剂变红的大肠杆菌区分开来，过夜培养后再次在麦康凯平板上挑取无色或浅橙色单菌落于LB液体培养基中增菌培养，使用五对靶向鉴定不同沙门氏菌的特异性PCR引物(表6)进行PCR扩增，琼脂糖凝胶电泳和观察鉴定结果；程序二：将各个脏器组织和浸泡脏器组织的缓冲液100μL涂布于麦康凯平板培养基上，过夜培养后挑取疑似无色或浅橙色单菌落于XLD上划线分离培养，过夜培养后挑取疑似粉红色单菌落于LB液体培养基中，增菌培养后使用五对靶向鉴定不同沙门氏菌的引物(表6)进行PCR扩增，琼脂糖凝胶电泳和观察鉴定结果。
表5：采集脏器及编号列表
Figure PCTCN2020140030-appb-000007
表6靶向鉴定不同沙门氏菌的的五对特异PCR引物
Figure PCTCN2020140030-appb-000008
通过上述两种程序的检测方法对70日龄40只种鸡中鸡白痢沙门氏菌带菌状况进行了系统的分离鉴定，40只鸡中每一只鸡对应各脏器样本如果能分离出鸡 白痢沙门氏菌，则判定为该鸡为鸡白痢沙门氏菌感染阳性。检测结果显示40只鸡中有9只鸡中分离出沙门氏菌，血清型都为鸡白痢沙门氏菌。试验中，同时使用商品化鸡白痢/鸡伤寒染色凝集抗原和惰性载体检测系统分别对每只鸡的血清进行了检测和对比分析，比对结果显示：①检出率方面，鸡白痢沙门氏菌细菌分离鉴定的结果中检出率为22.5％，商品化凝集抗原的检出率为50％，惰性载体检测系统S9-P的检出率为25％，惰性载体检测系统S9-P与细菌分离鉴定的结果检出符合率更好，符合率大于等于90％；②在细菌分离阴性样本，惰性载体检测系统S9-P检出4份阳性，分析考虑细菌分离鉴定过程中存在漏检的情况，也不排除使用抗菌素而导致分离漏检；③细菌分离阳性样本，惰性载体检测系统S9-P有3份为阴性，分析考虑少许数量的70日龄鸡在该年龄存在抗体效价偏低而导致检测漏检，也不排除在细菌感染的窗口期，没有产生超过检测限的抗体。
实施例6惰性载体间接凝集试验检测系统S9-P现场鸡全血检测和比较分析
使用惰性载体检测系统S9-P对A种鸡群现场随机检测148份全血，现场待检鸡采集5μL～10μL全血与5μL～10μL不同凝集诊断抗原充分混合，在2min之内判定结果。S9-P结果为阳性89份(60.14％)，阴性59份(39.86％)，测试结果表明该A种鸡群鸡白痢沙门氏菌感染阳性率高。而使用商品化凝集抗原同时和同步平行检测了19份，在19份平行检测样本中，商品化凝集抗原测试阳性结果13份，S9-P测试阳性结果19份，全为阳性，我们注意到商品化凝集抗原为阴性的6份样本中，S9-P检出5份阳性。本次测试中，商品化凝集抗原检测全血样本时阳性结果呈现的凝集反应微弱(图9)，表现凝集颗粒都为较细小的颗粒，且出现凝集反应阳性的时间较长。而S9-P在检测时出现阳性反应迅速，且强阳性反应凝集颗粒为较大块的凝集颗粒，清晰易判(图9)。
实施例7 Western blot验证惰性载体间接凝集试验检测系统S9-P检测结果
使用常规方法纯化提取S9-P菌株的P因子蛋白，分别纯化提取S9-P检测为阳性血清、阴性血清的IgG，以S9-P检测阳性血清、阴性血清IgG为一抗，羊抗鸡lgG-HRP为二抗进行Western blot实验，同时设立鸡白痢阳性血清IgG和SPF鸡血清IgG为一抗对照。检测结果显示，S9-P检测的阳性血清IgG识别P抗原因子，而S9-P检测的阴性血清IgG不能识别P抗原因子，未见到目的条带(图10)。S9-P检测的阳性血清，包括对照组为鸡白痢沙门氏菌人工感染SPF鸡血清，实验组1和实验组2来自临床种鸡血清(S9-P检测为阳性)；S9-P检测的阴性血清，包括对照组为SPF鸡血清，实验组1和实验组2来自临床种鸡血清(S9-P检测为阴性)。
实施例8惰性载体间接凝集试验检测系统S9-P菌株敏感性测试
使用鸡伤寒沙门氏菌SG01分别在5日龄、19日龄感染SPF鸡，使用鸡白痢沙门氏菌SP03感染34日龄SPF鸡，使用惰性检测系统S9-P菌株检测不同感染时间的SPF鸡。结果显示抗体增长符合一般规律：①5日龄SPF鸡人工感染鸡伤寒沙门氏菌，在第一周(第7天，12日龄鸡)测不出感染抗体；在第二周(第14天，19日龄鸡)开始，已能测出鸡群部分个体的感染抗体(阳性率5.88％，1/17)；②感染鸡白痢/鸡伤寒沙门氏菌后的第43天能全部测出感染鸡群个体的抗体；③未攻毒组SPF阳性率一直保持0％。
综上所述，本发明为一种新颖的惰性载体间接凝集试验检测系统S9-P，其创新和优势体现在：现有的鸡白痢沙门氏菌商品化凝集抗原采用的是鸡白痢标准 株(或/和变异株)全菌灭活制备凝集抗原，理论上和实践中都证明了这种凝集抗原存在交叉非特异凝集反应，敏感性不高，稳定性和重复性不佳的瓶颈制约。本发明涉及的新型惰性载体检测系统S9-P的研发基于前期筛选并验证与各种背景鸡血清无交叉凝集反应的惰性载体(菌)S9，避免了多成分全菌抗原造成的非特异性交叉凝集反应干扰。本发明同时发现并验证了抗原因子P仅存在表达于鸡白痢沙门氏菌种内且高度保守，首创性使用基于惰性载体(菌)S9菌体表面表达和携带单一抗原因子P，构建新型惰性载体检测系统S9-P，特异性靶向鸡白痢沙门氏菌感染全血或血清，能够实现圈栏旁鸡舍采集测试鸡一滴血(10μL左右)，通过肉眼观察和2分钟内现场快速判定凝集反应结果，监测、检测鸡群中鸡白痢沙门氏菌感染，特异、敏感。本发明创新性改进和最大完善现有凝集抗原抗体检测的多年来一直使用(自上世纪50年代以来)的传统、经典凝集试验的特异性和敏感性(不稳定、重复性不佳)技术瓶颈制约，具有很好的应用价值和市场前景。

Claims (9)

  1. 惰性载体间接凝集试验检测系统，其特征在于，所述间接凝集试验检测系统用于检测鸡白痢沙门氏菌感染，所述间接凝集试验检测系统包括惰性载体菌S9和在其表面展呈表达和携带P抗原因子的复合体S9-P，所述间接凝集试验检测系统是含有鸡白痢沙门氏菌p基因的惰性载体菌S9，惰性载体菌S9的保藏编号为CGMCC No.17340，所述基因p的获得是以鸡白痢沙门氏菌CVCC526株参考株基因组DNA为模板，通过设计特异性引物对进行PCR扩增得到目的基因p，所述特异性引物对为：
    p-UP：5'-ATG AAA CGT TCA CTT ATT GCT GCT-3'
    p-LO:5'-TTA A TCA GTT AAT ACC GTC ATC GTC AG-3'。
  2. 根据权利要求1所述的惰性载体间接凝集试验检测系统，其特征在于，所述检测系统是通过将鸡白痢沙门氏菌p基因与pBR322质粒连接后导入S9电转化感受态细胞中鉴定正确的菌株。
  3. 权利要求1所述的惰性载体间接凝集试验检测系统的构建方法，其特征在于，包括以下步骤：
    1)鸡白痢沙门氏菌中P抗原因子编码基因p的获得；
    2)鸡白痢沙门氏菌中P抗原因子编码基因p与pBR322质粒的连接获得重组质粒；
    3)重组质粒p-pBR322转化入S9电转化感受态细胞鉴定得到重组菌株即为惰性载体间接凝集试验检测系统S9-P。
  4. 根据权利要求3所述的惰性载体间接凝集试验检测系统的构建方法，其特征在于，所述步骤1)中基因p的获得是以鸡白痢沙门氏菌CVCC526株参考株基因组DNA为模板，通过设计特异性引物对进行PCR扩增得到目的基因p。
  5. 根据权利要求4所述的惰性载体间接凝集试验检测系统的构建方法，其特征在于，所述特异性引物对为：
    p-UP：5'-ATG AAA CGT TCA CTT ATT GCT GCT-3'
    p-LO:5'-TTA A TCA GTT AAT ACC GTC ATC GTC AG-3'。
  6. 权利要求1～2任一项所述的惰性载体间接凝集试验检测系统在制备抗原或抗体的间接凝集反应试剂或试剂盒或鸡相关病原感染检测用试剂或试剂盒中的应用。
  7. 权利要求1～2任一项所述的惰性载体间接凝集试验检测系统在制备鸡白痢沙门氏菌感染检测用试剂或试剂盒中的应用。
  8. 一种鸡白痢沙门氏菌感染检测试剂盒，其特征在于，所述检测试剂盒包括权利要求1～2任一项所述的惰性载体间接凝集试验检测系统。
  9. 根据权利要求8所述的检测试剂盒，其特征在于，所述检测试剂盒还包括阳性对照和阴性对照。
PCT/CN2020/140030 2020-05-12 2020-12-28 惰性载体间接凝集试验检测系统及其应用 WO2021227514A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
GB2201199.3A GB2600848B (en) 2020-05-12 2020-12-28 Inert bio-vector indirect agglutination test detection system and uses thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010400163.4A CN111537712B (zh) 2020-05-12 2020-05-12 惰性载体间接凝集试验检测系统及其应用
CN202010400163.4 2020-05-12

Publications (1)

Publication Number Publication Date
WO2021227514A1 true WO2021227514A1 (zh) 2021-11-18

Family

ID=71977642

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/140030 WO2021227514A1 (zh) 2020-05-12 2020-12-28 惰性载体间接凝集试验检测系统及其应用

Country Status (3)

Country Link
CN (1) CN111537712B (zh)
GB (1) GB2600848B (zh)
WO (1) WO2021227514A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117491657A (zh) * 2023-11-03 2024-02-02 中国兽医药品监察所 鸡毒支原体阳性血清和阴性血清标准物质及制备方法

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111537712B (zh) * 2020-05-12 2021-01-05 扬州大学 惰性载体间接凝集试验检测系统及其应用
CN112481287B (zh) * 2020-11-17 2022-01-28 扬州大学 一种表达sef14功能性菌毛的重组菌及其应用
CN116715737B (zh) * 2022-11-24 2024-02-23 扬州大学 新型冠状病毒受体结合域B细胞表位的靶标抗原及其在Peg菌毛展呈表达及应用
CN116769801A (zh) * 2023-06-16 2023-09-19 扬州大学 基于啮齿类柠檬酸杆菌eae基因的惰性载体菌的重组表达系统及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8771936B2 (en) * 2008-12-24 2014-07-08 Cj Cheiljedang Corporation Bacteriophage and antibacterial composition comprising the same
CN104789500A (zh) * 2015-04-03 2015-07-22 江苏省家禽科学研究所 一种鸡白痢染色凝集抗原及其制备方法和应用
CN109207417A (zh) * 2018-09-19 2019-01-15 湖北省农业科学院畜牧兽医研究所 鸡白痢-j亚群禽白血病双重平板凝集抗原及其制备方法
CN110218668A (zh) * 2019-05-21 2019-09-10 扬州大学 一种惰性载体沙门氏菌及其潜在应用
CN111537712A (zh) * 2020-05-12 2020-08-14 扬州大学 惰性载体间接凝集试验检测系统及其应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107446851B (zh) * 2017-08-25 2020-06-23 扬州大学 一种鸡白痢凝集抗原及其制备方法
CN110257276B (zh) * 2019-05-21 2020-03-24 扬州大学 一种惰性载体大肠杆菌及其潜在应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8771936B2 (en) * 2008-12-24 2014-07-08 Cj Cheiljedang Corporation Bacteriophage and antibacterial composition comprising the same
CN104789500A (zh) * 2015-04-03 2015-07-22 江苏省家禽科学研究所 一种鸡白痢染色凝集抗原及其制备方法和应用
CN109207417A (zh) * 2018-09-19 2019-01-15 湖北省农业科学院畜牧兽医研究所 鸡白痢-j亚群禽白血病双重平板凝集抗原及其制备方法
CN110218668A (zh) * 2019-05-21 2019-09-10 扬州大学 一种惰性载体沙门氏菌及其潜在应用
CN111537712A (zh) * 2020-05-12 2020-08-14 扬州大学 惰性载体间接凝集试验检测系统及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
YANG WEIFENG: "Development of Monoclonal Antibody Against Salmonella PEG Fimrbrae and Preliminary Clinical Application", CHINESE MASTER’S THESES FULL-TEXT DATABASE, AGRICULTURE SCIENCE AND TECHNOLOGY, no. 2, 15 February 2017 (2017-02-15), XP055866946 *
YUE MIN, RANKIN SHELLEY C., BLANCHET RYAN T., NULTON JAMES D., EDWARDS ROBERT A., SCHIFFERLI DIETER M.: "Diversification of the Salmonella Fimbriae: A Model of Macro- and Microevolution", PLOS ONE, vol. 7, no. 6, 12 June 2012 (2012-06-12), pages e38596, XP055866945, DOI: 10.1371/journal.pone.0038596 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117491657A (zh) * 2023-11-03 2024-02-02 中国兽医药品监察所 鸡毒支原体阳性血清和阴性血清标准物质及制备方法

Also Published As

Publication number Publication date
GB2600848A (en) 2022-05-11
GB2600848B (en) 2024-06-26
CN111537712B (zh) 2021-01-05
GB202201199D0 (en) 2022-03-16
CN111537712A (zh) 2020-08-14

Similar Documents

Publication Publication Date Title
WO2021227514A1 (zh) 惰性载体间接凝集试验检测系统及其应用
Kazakova et al. Recombinant protein p30 for serological diagnosis of African swine fever by immunoblotting assay
US12085566B2 (en) Inert carrier Salmonella and potential use thereof
WO2020233147A1 (zh) 一种惰性载体大肠杆菌及其潜在应用
WO2021232799A1 (zh) 一种泛性型惰性载体沙门氏菌及其潜在应用
CN113956362B (zh) 一种重组猫细小病毒vp2蛋白抗原及其在抗体诊断和疫苗制备中的应用
CN105755118B (zh) 免疫磁珠环介导等温扩增法快速检测副溶血性弧菌的方法
CN111830257A (zh) 一种猪源胞内劳森菌ipma抗原检测方法及其应用
Yang et al. Self-made Salmonella Pullorum agglutination antigen development and its potential practical application
CN106443015A (zh) 一种基于hexon蛋白N端保守区的检测禽腺病毒抗体的ELISA试剂盒
CN113684189A (zh) 一株鸡新型圆圈病毒3型毒株及基于该病毒的检测体系
WO2021232800A1 (zh) 一种泛性型惰性载体大肠杆菌及其潜在应用
CN112940089B (zh) 胞内劳森菌flgE重组蛋白及胞内劳森菌抗体检测试剂盒
US20230375548A1 (en) Indirect enzyme-linked immunosorbent assay detection kit based on p30 protein and p22 protein of african swine fever virus
CN109521199B (zh) 一种用于检测禽白血病病毒的胶体金检测试剂盒及其应用
CN108559754A (zh) 一种猪繁殖与呼吸综合征病毒gp5基因修饰的重组蛋白及其应用
CN107664694A (zh) 一种基于e2蛋白检测猪非典型瘟病毒抗体的elisa试剂盒
CN117229421B (zh) 非洲猪瘟病毒外囊膜蛋白CD2v胞外区靶标抗原特定表位及其抗体和应用
CN113045673A (zh) 一种基于嵌合p54抗原表位的非洲猪瘟病毒抗体检测试剂盒及应用
Longyant et al. Specific monoclonal antibodies raised against Taura syndrome virus (TSV) capsid protein VP3 detect TSV in single and dual infections with white spot syndrome virus (WSSV)
CN116715737B (zh) 新型冠状病毒受体结合域B细胞表位的靶标抗原及其在Peg菌毛展呈表达及应用
CN117186190B (zh) 非洲猪瘟病毒外囊膜蛋白p12靶标表位抗原及其在早期检测诊断中的应用
CN115785286B (zh) 一种猪繁殖与呼吸综合征病毒的融合蛋白及其应用
CN114106158B (zh) 一种靶向猪伪狂犬病毒gD蛋白的纳米抗体、制备方法和应用
CN116375823B (zh) 携带幽门螺杆菌毒力因子的b细胞表位靶标抗原及其表达载体和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20935411

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 202201199

Country of ref document: GB

Kind code of ref document: A

Free format text: PCT FILING DATE = 20201228

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20935411

Country of ref document: EP

Kind code of ref document: A1