Biomarker for detecting riemerella anatipestifer and detection method

Abstract

The invention relates to the technical field of riemerella anatipestifer detection, and provides a biomarker and a detection method for detecting riemerella anatipestifer, and the biomarker is a host mucosal lesion product generated by induction of specific virulence factors of riemerella anatipestifer after duck infection. Comprising a mucoprotein cracking fragment containing a linear epitope GTTPSPVP, a sialylated sugar chain containing a specific sialic acid modification motif, and a Muc2 degradation peptide fragment. The detection method comprises the following steps: collecting a sample, pretreating, detecting corresponding markers for excrement, respiratory tract lavage fluid and mucous membrane scrape samples by adopting ELISA (Enzyme-Linked Immunosorbent Assay), an immunochromatography test strip and a lectin chip respectively, and judging a result according to a set threshold value. The kit is high in specificity, covers the whole infection period, can realize early and on-site rapid detection, and improves the diagnosis reliability.

Description

Technical Field

[0001] This invention relates to the field of Riemerella anatipestifer detection technology, specifically to biomarkers and detection methods for Riemerella anatipestifer. Background Technology

[0002] Riemerella anatipestifer is a Gram-negative bacillus, a major pathogen that specifically infects waterfowl, primarily causing infectious serositis in ducks, geese, and other poultry. The disease is characterized by fibrinous pericarditis, perihepatitis, and meningitis; it has a rapid onset and spreads quickly, leading to a mortality rate of nearly 30% in ducklings. Survivors experience stunted growth, causing severe economic losses to the poultry industry. Therefore, rapid and accurate detection of Riemerella anatipestifer is crucial for early prevention and control of outbreaks and minimizing economic losses.

[0003] Current detection technologies primarily rely on direct pathogen detection methods, such as bacterial isolation and culture, polymerase chain reaction (PCR), and antigen-antibody detection. However, these methods have significant limitations: bacterial culture is time-consuming and susceptible to interference from other bacteria in the sample; while PCR technology has high sensitivity, it is prone to false negatives in the early stages of infection because bacterial proliferation lags behind the host's pathological response; and antibody detection based on bacterial surface antigens is easily affected by pathogen antigen variation, leading to false negatives or fluctuations in sensitivity. Furthermore, traditional methods struggle to distinguish between active infection and latent carrier states, have poor adaptability to highly mucous samples, and complex matrices often interfere with detection signals. More critically, current technologies cannot capture host-specific response signals in the early stages of infection. *Riemerella anatipestifer* directly damages the host mucosa through virulence factors such as sialidase and metalloproteinases, inducing the production of characteristic biomarkers, but these host-directed molecular scars have not been effectively developed and utilized. Current methods lack specific probes and suitable detection platforms for these biomarkers, hindering early, highly specific diagnosis. Simultaneously, there is a gap in on-site rapid detection technologies. Farms urgently need simple instruments and screening tools that can produce results within 10 minutes. However, existing rapid testing products lack sensitivity for low-concentration biomarkers and are susceptible to non-specific adsorption interference from mucus samples. The sample pretreatment process also lacks a unified approach, failing to simultaneously protect the three types of biomarkers from endogenous protease degradation, resulting in low biomarker recovery rates and poor result stability. These technical bottlenecks make it difficult for existing testing systems to cover the entire infection cycle and provide full-chain support for precise prevention and control.

[0004] Therefore, it is urgent to establish a new detection system to break through the limitations of traditional methods in terms of timeliness, specificity, and applicability, and fundamentally solve core problems such as early missed detection, misdiagnosis of variants, insufficient on-site resources, and sample interference. Summary of the Invention

[0005] To address the problems existing in the background art, the present invention provides a biomarker for detecting Riemerella anatipestifer infection. The biomarker is a host mucosal damage product induced by Riemerella anatipestifer specific virulence factors after duck infection, and is selected from any of the following: Characteristic mucin cleavage fragments shed from intestinal or respiratory mucosal cells, with an isoelectric point pI of 4.5–5.2, are produced by cleavage of host Muc5ac protein by sialidase or metalloproteinase of Riemerella anatipestifer, and their sequence SEQ ID NO:2 contains the linear epitope GTTPSPVP. The aberrantly expressed sialylated glycan structure contains a terminal α2,6-linked sialic acid-modified N-acetylgalactosamine GalNAc-β1,3-Gal motif; The sequence of the carboxyl-terminal degradation peptide of Muc2 protein, specifically secreted by goblet cells in the respiratory tract, is shown in SEQ ID NO:1.

[0006] This invention provides a kit for detecting Riemerella anatipestifer infection, comprising a sample pretreatment solution, a TMB substrate solution, a signal intensity colorimetric card, and: The mucin cleavage fragment detection module includes an ELISA plate coated with anti-Muc5ac monoclonal antibody and biotin-labeled anti-GTTPSPVP monoclonal antibody; The sialylated glycan detection module includes a glass slide chip with immobilized lectin MAL-II and Cy5 fluorescently labeled streptavidin; The Muc2 degradation peptide detection module includes an immunochromatographic test strip containing anti-Muc2 polyclonal antibody and goat anti-rabbit IgG coated on a nitrocellulose membrane.

[0007] Furthermore, the heavy chain CDR3 sequence of the anti-GTTPSPVP monoclonal antibody is CASRGPYFDYW (SEQ ID NO:3), and the light chain CDR3 sequence is CQQYRTYPHVF (SEQ ID NO:4); the enzyme-labeled plate coating buffer is carbonate buffer at pH 9.6.

[0008] Furthermore, in the sialylated glycan detection module, the immobilized lectin MAL-II specifically binds to the sialylic acid-modified N-acetylgalactosamine GalNAc-β1,3-Gal motif linked at the terminal α2,6-; and Cy5 fluorescently labeled streptavidin detects the biotin-labeled lectin-glycan complex.

[0009] Furthermore, in the Muc2 degradation peptide detection module, the anti-Muc2 polyclonal antibody specifically recognizes the peptide segment of SEQ ID NO:1; the goat anti-rabbit IgG is the quality control line of the test strip.

[0010] Furthermore, the sample pretreatment solution contained 50 mM Tris-HCl buffer, 1% Triton X-100, 5 mM EDTA, and a 10 μg / mL mixture of protease inhibitors.

[0011] This invention provides a method for detecting Riemerella anatipestifer infection, comprising the following steps: S1, Sample collection: Obtain duck feces, respiratory lavage fluid, or mucosal scrapings as samples; S2, use pretreatment solution to process the sample; S3: For fecal samples, use step S31; for respiratory lavage fluid samples, use step S33 or S31; for mucosal scraping samples, use step S32; Perform biomarker detection: S31, Detection and quantification of mucin cleavage fragments: S311, add the pretreated sample to an ELISA plate coated with anti-Muc5ac monoclonal antibody and incubate; S312, incubated with biotin-labeled anti-GTTPSPVP monoclonal antibody; S313, with the addition of streptavidin-HRP conjugate and TMB substrate for color development; S314, Quantitatively calculate the concentration of mucin cleavage fragments based on a standard curve, wherein the standard curve is established using a gradient dilution of recombinant protein containing the GTTPSPVP epitope; S32, Detection of sialylated glycan chains: S321, the pretreated sample is dropped onto the glass slide chip with immobilized MAL-II lectin; S322, incubated with Cy5 fluorescently labeled streptavidin; S323, scan fluorescence signals and calculate lectin binding values; Detection and concentration quantification of S33 and Muc2 degradation peptides: S331, Load the pretreated sample into the sample application area of ​​the immunochromatographic test strip; S332, through chromatography, the peptide shown in SEQ ID NO:1 in the sample binds to the coated anti-Muc2 polyclonal antibody; S333, based on the ratio of the color intensity of the test line to the control line on the test strip, the concentration of Muc2 degradation peptides is quantified with reference to the colorimetric card; S334, validation of the validity of the sheep anti-rabbit IgG quality control line results; S4. A positive result for Riemerella anatipestifer infection is determined when the test value meets any of the following conditions: Concentration of mucin cleavage fragments > 15 ng / mL; Sialized glycan binding value > 3 times that of negative control; The concentration of Muc2-degraded peptides is > 8 ng / mL.

[0012] The beneficial effects achieved by this invention are as follows: First, this invention provides three host mucosal damage products specifically produced after infection with Riemerella anatipestifer as biomarkers, including characteristic mucin cleavage fragments shed from intestinal or respiratory mucosal cells, abnormally expressed sialylated glycan structures, and carboxyl-terminal degradation peptides of Muc2 protein specifically secreted by respiratory goblet cells. These biomarkers are specifically induced by bacterial virulence factors such as sialylase, metalloproteinases, and serine proteases, rather than ordinary inflammatory response products, and their detection is highly specific. The exposure of linear epitopes in the mucin cleavage fragments and the rare modification of sialylated glycans enhance signal stability, overcoming the limitations of traditional detection methods at low bacterial loads and achieving efficient capture of early infection.

[0013] Second, the kit design includes three complementary detection modules: the mucin cleavage fragment detection module uses double antibody sandwich ELISA technology, the sialylated glycan detection module is based on the lectin chip platform, and the Muc2 degradation peptide detection module uses immunochromatographic test strips. The multi-module design achieves comprehensive coverage of different sample types and detection scenarios. The mucin module is suitable for quantitative analysis of high-volume samples such as feces, the glycan module shows strong interference resistance in high-mucus respiratory samples, and the test strip module supports rapid on-site screening, ensuring the flexibility and applicability of the test. Third, based on the three biomarkers, this invention designs a detection method for Riemerella anatipestifer. The sample pretreatment solution contains specific buffers, surfactants, chelating agents, and protease inhibitors, effectively protecting the integrity of the biomarkers and inhibiting endogenous enzymatic digestion. Simultaneously, the high-affinity CDR3 design of the antibody and the optimized immobilization of the lectin significantly reduce non-specific binding and background interference, improving the sensitivity and accuracy of the detection. This ensures the stability of the biomarkers in complex samples, guaranteeing the reliability and reproducibility of the results. This invention employs a multi-biomarker joint decision-making strategy; a positive infection result is determined when any biomarker's detection value exceeds a threshold, improving diagnostic reliability, avoiding the false negative risk of single methods, and covering different stages of infection, from early enzymatic cleavage events to glycosylation modification in the progression phase and residual peptides in the recovery phase, achieving effective monitoring of the entire infection cycle. This comprehensive detection scheme significantly enhances the diagnostic efficacy of Riemerella anatipestifer, providing a more comprehensive solution for farms and laboratories. Attached Figure Description

[0014] Figure 1 This is a flowchart of the detection method for detecting Riemerella anatipestifer of the present invention. Detailed Implementation

[0015] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. In addition, the forms of the various structures described in the following embodiments are merely illustrative. The present invention is not limited to the structures described in the following embodiments. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Reference Figure 1 First, let's explain each sequence in detail: SEQ ID NO:1 is a Muc2 degradation peptide with the sequence: Pro-Arg-Gly-Asp-Thr-Val-Val-Val-Pro-Cys, or PRGDTVVVPC. It is a C-terminal degradation peptide of the Muc2 protein secreted by respiratory goblet cells and produced by protease cleavage of Riemerella anatipestifer. As a specific biomarker, the immunochromatographic test strip uses an anti-Muc2 polyclonal antibody to directly recognize this sequence.

[0017] SEQ ID NO:2 is an epitope sequence in a mucin cleavage fragment. The core epitope is GTTPSPVP, which is located within the mucin cleavage fragment. This fragment is the product of Muc5ac protein being cleaved by bacterial sialidase / metalloproteinase and contains the linear epitope GTTPSPVP. This epitope is specifically bound to a biotinylated anti-GTTPSPVP monoclonal antibody.

[0018] SEQ ID NO:3 is the heavy chain CDR3 of the anti-GTTPSPVP antibody, with the amino acid sequence CASRGPYFDYW. This is the complementarity-determining region 3 (CDR3) of the heavy chain of the anti-GTTPSPVP monoclonal antibody, which determines the antibody's specific recognition ability of the antigen epitope GTTPSPVP. The terminal W (tryptophan) and Y (tyrosine) residues are key residues for antigen binding.

[0019] SEQ ID NO:4 is the light chain CDR3 of the anti-GTTPSPVP antibody, with the amino acid sequence CQQYRTYPHVF. It pairs with SEQ ID NO:3 to form the light chain CDR3 region of the same antibody, together forming an antigen-binding pocket. It contains hydrophobic residues such as Y (tyrosine), H (histidine), and F (phenylalanine), which have strong binding affinity to epitopes.

[0020] Table 1 Key sequences and their functions

[0021] sequence Corresponding entity effect SEQ ID NO:1 Muc2 degradation peptides (10 peptides) Respiratory infection-specific markers, recognized by anti-Muc2 antibodies SEQ ID NO:2 Mucin cleavage fragments (containing GTTPSPVP epitopes) Intestinal / respiratory mucosal injury markers were captured by anti-GTTPSPVP antibody. SEQ ID NO:3 Anti-GTTPSPVP antibody heavy chain CDR3 Endow antibodies with the ability to specifically bind to antigenic epitopes. SEQ ID NO:4 Anti-GTTPSPVP antibody light chain CDR3 Working in conjunction with heavy chain CDR3 to complete antigen recognition

[0022] This invention provides a biomarker for detecting *Riemerella anatipestifer* infection. The biomarker is a host mucosal damage product induced by *Riemerella anatipestifer*-specific virulence factors after duck infection, selected from any of the following: characteristic mucin cleavage fragments shed from intestinal or respiratory mucosal cells, with an isoelectric point (pI) of 4.5–5.2, wherein the characteristic mucin cleavage fragments are generated by *Riemerella anatipestifer* sialidase or metalloproteinase cleaving the host Muc5ac protein, and its sequence SEQ ID NO:2 contains the linear epitope GTTPSPVP; an aberrantly expressed sialylated glycan structure containing a terminal α2,6-linked sialic acid-modified N-acetylgalactosamine GalNAc-β1,3-Gal motif; and a carboxyl-terminal degradation peptide of Muc2 protein specifically secreted by respiratory goblet cells, with the sequence shown in SEQ ID NO:1.

[0023] The mucin cleavage fragment (marker a) has a molecular weight of 35–45 kDa and an isoelectric point (pI) of 4.5–5.2, with the core epitope containing GTTPSPVP (SEQ ID NO:2). The formation mechanism involves *Rimeria anatidae* secreting sialidase / metalloproteinase, which specifically cleaves the O-glycosylated region of the host respiratory / intestinal Muc5ac protein, exposing the hydrophobic linear epitope GTTPSPVP. This epitope is masked by glycans in intact mucin; cleavage leads to deglycosylation and a decrease in pI (increased acidity). It exhibits high specificity, appearing only after infection and undetectable in healthy tissues; enzyme cleavage precedes bacterial proliferation, overcoming the bottleneck of detection in low-load samples.

[0024] The sialylated glycan chain (marker b) contains a GalNAc-β1,3-Gal motif modified with sialic acid (Neu5Ac) at the terminal α2,6-linked end (structural formula: Neu5Acα2-6GalNAcβ1-3Gal). The formation mechanism involves bacterial virulence factors activating the host sialyltransferase ST6GalNAc1, which adds α2,6-sialic acid to the T antigen of mucin (Galβ1-3GalNAc), generating an Sda-like structure. This modification is competitively inhibited by α2,3-sialylation in the mucosa of healthy ducks. The α2,6 bond is resistant to sialylase degradation, and the signal is stable; only MAL-II (non-SNA) can bind with high affinity.

[0025] The Muc2 degradation peptide (marker c) sequence Pro-Arg-Gly-Asp-Thr-Val-Val-Val-Pro-Cys (SEQ ID NO:1) has a molecular weight of approximately 1.2 kDa. The formation mechanism involves the bacterial serine protease RapA specifically cleaving the C-terminus of the Muc2 protein secreted by goblet cells, exposing the RGD integrin-binding domain (Arg-Gly-Asp) and the hydrophobic core (Val / Pro enrichment region). The C-terminal cysteine ​​residue (Cys) stabilizes the conformation via a disulfide bond. It exhibits cell-specific characteristics, being expressed only in respiratory goblet cells; short peptide-tolerant samples show proteolytic hydrolysis.

[0026] The core component of the mucin cleavage fragment detection module is anti-Muc5ac monoclonal antibody (targeting the intact protein framework); the detection antibody is biotinylated anti-GTTPSPVP monoclonal antibody (targeting the neonatal epitope SEQ ID NO:2). In the heavy chain CDR3 (SEQ ID NO:3), arginine (R...) in CASRGPYFDYW... 5 ), tyrosine (Y) 7 ) forms hydrogen bonds with threonine (T) and proline (P) at the epitope; the light chain CDR3 (SEQ ID NO:4): CQQYRTYPHVF contains histidine (H) 8 ), phenylalanine (F1) 0 It locks valine (V) through hydrophobic interactions.

[0027] The core component of the sialylated glycan detection module is the immobilized lectin MAL-II (Maackia amurensislectin II), cross-linked to an APTES-activated slide via glutaraldehyde; and Cy5-streptavidin, which binds to biotin-labeled glycans. The β-trefoil lectin domain of MAL-II specifically recognizes the sialic acid carboxyl group and GalNAc acetylamino group in α2,6-Sia-GalNAc, with a binding constant KD ≤ 10⁻. 8 M. 200 μm dot pitch increases detection throughput; Cy5 quantum efficiency is 3 times that of FITC.

[0028] The core component of the Muc2 degradation peptide detection module is anti-SEQ ID NO:1 polyclonal antibody (targeting RGD and the hydrophobic core); the quality control line is goat anti-rabbit IgG (to verify the activity of the labeled antibody). Blocking with 0.1% BSA + 5% sucrose eliminates non-specific adsorption of mucus; the 8 μm pore size of the nitrocellulose membrane optimizes the chromatography speed (≤10 minutes); the T / C colorimetric intensity ratio enables semi-quantitative analysis, suitable for rapid on-site testing.

[0029] The sample pretreatment solution is as follows: Table 2. Composition and Function of Sample Pretreatment Solution

[0030] Components concentration Function Tris-HCl buffer 50 mM (pH 8.0) Maintaining the optimal pH for proteases Triton X-100 1% (v / v) Markers of mucosal cell lysis EDTA 5 mM Chelating metal ions (a marker of inhibition of metalloproteinase degradation) Sialidase inhibitors 10 mM Blocking α2,3-sialidase activity (preventing demodification of glycan markers) Protease inhibitor mixture 10 μg / mL Contains AEBSF (serine protease inhibitor) and E-64 (cysteine ​​protease inhibitor).

[0031] Table 3. Complementarity of the combined detection of three biomarkers

[0032] Sample type Optimal module reason Early infection Mucin fragments Enzyme digestion occurs before bacterial proliferation. High-viscosity samples sugar chain lectins are resistant to mucin interference On-site rapid testing Muc2 peptide segment The test strips produce results in 10 minutes.

[0033] The biomarker design targets pathogen-specific induced products (non-general inflammatory biomarkers); the enzyme cleavage epitopes (a), rare sugar modifications (b), and protease cleavage peptides (c) are all irreversible.

[0034] This invention provides a kit for detecting Riemerella anatipestifer infection, comprising a sample pretreatment solution, a TMB substrate solution, a signal intensity colorimetric card, and a mucin cleavage fragment detection module, including an ELISA plate coated with anti-Muc5ac monoclonal antibody and biotin-labeled anti-GTTPSPVP monoclonal antibody; a sialylated glycan detection module, including a glass slide chip immobilized with lectin MAL-II and Cy5 fluorescently labeled streptavidin; and a Muc2 degradation peptide detection module, including an immunochromatographic test strip containing anti-Muc2 polyclonal antibody and goat anti-rabbit IgG coated on a nitrocellulose membrane.

[0035] The heavy chain CDR3 sequence of the anti-GTTPSPVP monoclonal antibody is CASRGPYFDYW (SEQ ID NO:3), and the light chain CDR3 sequence is CQQYRTYPHVF (SEQ ID NO:4); the ELISA plate coating buffer is pH 9.6 carbonate buffer. In the sialylated glycan detection module, immobilized lectin MAL-II specifically binds to the α2,6-terminal sialic acid-modified N-acetylgalactosamine GalNAc-β1,3-Gal motif; Cy5 fluorescently labeled streptavidin detects the biotin-labeled lectin-glycan complex. In the Muc2 degradation peptide detection module, the anti-Muc2 polyclonal antibody specifically recognizes the peptide sequence of SEQ ID NO:1; the goat anti-rabbit IgG is the control line of the test strip; the sample pretreatment solution contains 50 mM Tris-HCl buffer, 1% Triton X-100, 5 mM EDTA, and a 10 μg / mL protease inhibitor mixture.

[0036] The mucin cleavage fragment detection module comprises an ELISA plate coated with anti-Muc5ac monoclonal antibody and a biotinylated anti-GTTPSPVP monoclonal antibody. The anti-Muc5ac monoclonal antibody acts as a capture antibody, recognizing the intact backbone structure of mucin cleavage fragments; the biotinylated anti-GTTPSPVP monoclonal antibody targets the linear epitope GTTPSPVP (SEQ ID NO:2) exposed after bacterial cleavage, forming a double-antibody sandwich complex. This module shares the sample pretreatment solution with the sialylated glycan detection module but performs ELISA independently, providing a quantitative gold standard for high-load samples (such as intestinal mucus).

[0037] The sialylated glycan detection module consists of a glass slide chip immobilized with lectin MAL-II and Cy5-labeled streptavidin. Lectin MAL-II specifically binds to the terminal α2,6-sialic acid modification motif (Neu5Acα2-6GalNAcβ1-3Gal) in marker b; Cy5-streptavidin amplifies the fluorescence signal via a biotin-avidin system, achieving high-sensitivity detection. The detection results can be cross-validated with the mucin cleavage fragment module, making it particularly suitable for high-mucus respiratory samples (lectin is resistant to mucus interference).

[0038] The Muc2 degradation peptide detection module is an immunochromatographic test strip. Its nitrocellulose membrane is coated with anti-SEQ ID NO:1 polyclonal antibody (detection line) and goat anti-rabbit IgG (control line). The anti-SEQ ID NO:1 antibody (recognizing the PRGDTVVVPC peptide) captures Muc2 degradation peptides in the sample; the goat anti-rabbit IgG binds to the labeled antibody to verify the effectiveness of the chromatography process. As a rapid detection solution, the Muc2 degradation peptide detection module complements the aforementioned two modules, providing precise and rapid detection, and is suitable for on-site screening.

[0039] The sample pretreatment solution contains Tris-HCl buffer, Triton X-100, EDTA, and a protease inhibitor, uniformly treating three types of samples to release and protect biomarkers. The TMB substrate solution is specifically designed for the ELISA colorimetric reaction of the mucin module. A signal intensity colorimetric card is used to standardize the interpretation of the T / C line colorimetric ratio on the test strips.

[0040] Table 4. Target locking mechanism of kits for detecting Riemerella anatipestifer infection.

[0041] Module Target structure Biological basis Mucin cleavage module GTTPSPVP epitope (SEQ ID NO:2) Newly generated linear epitopes exposed after bacterial virulence enzymes cleave host Muc5ac (glycosylated masking in healthy tissue). sugar chain module α2,6-Sia-GalNAcβ1-3Gal Infection activates the host's ST6GalNAc1 enzyme, which links sialic acid to GalNAc via α2,6 bonds (healthy tissues primarily use α2,3 bonds). Muc2 peptide module PRGDTVVVPC (SEQ ID NO:1) Bacterial proteases specifically cleave the C-terminus of the goblet cell Muc2 protein to release the RGD integrin-binding domain.

[0042] The heavy chain CDR3 (CASRGPYFDYW) and light chain CDR3 (CQQYRTYPHVF) of the anti-GTTPSPVP monoclonal antibody lock the epitope via hydrogen bonding / hydrophobic interactions. Only MAL-II can bind with high affinity to α2,6-Sia modifications (KD≤10⁻). 8(M), conventional lectin SNAs only recognize α2,3 linkages. Anti-Muc2 polyclonal antibodies strictly target SEQ ID NO:1, with a homologous sequence binding rate of <5%. EDTA in the pretreatment solution chelates metal ions (inhibiting metalloproteinases), and a sialidase inhibitor prevents glycan demodification. The sample pad on the test strip is blocked with 0.1% BSA + 5% sucrose to eliminate non-specific adsorption of mucus. The lectin chip has a spot spacing of 200 μm, and a single chip can detect 48 samples.

[0043] Table 5. Module synergistic effects covering the entire infection cycle and their mechanisms of action.

[0044] Infection stage Advantage Module Inspection mark Problem Solving Early stage (≤24h) Mucin module Enzyme digestion occurs before bacterial proliferation. Breaking the PCR detection limit (bacterial count <10³ CFU / mL) Progress period sugar chain module Glycosylation modification is irreversible Avoid false negatives in antibody tests (antigen variation) Recovery period Muc2 peptide module Short peptides have a long metabolic half-life (>72h). Monitoring treatment effectiveness

[0045] The mucin module (ELISA) combined with the glycan module (chip) provides dual validation with 100% sensitivity (n=50). The test strips produce results in 10 minutes, with a detection limit as low as 8 ng / mL, requiring no instrument. The pretreatment solution increases the detection rate of fecal samples from 70% to 98%.

[0046] This kit achieves a shift from detecting bacteria to capturing molecular scars caused by bacteria through host-guided virulence enzyme cleavage, biomarker release pathological mechanism analysis, irreversible structural locking of cleavage epitopes / rare sugar modification / cleavage peptides, detection tool-adaptive antibodies, CDR3 protection, lectin uniqueness, and test strip optimization. It achieves ELISA / chip / chromatographic complementarity, covering all scenarios; and multi-biomarker joint decision-making improves diagnostic reliability.

[0047] Example 1: The preparation process of anti-GTTPSPVP monoclonal antibody is as follows: Immunogen preparation involves covalently coupling a synthetic peptide containing the linear epitope GTTPSPVP (SEQ ID NO:2) to the carrier protein keyhole hemocyanin (KLH) via a carbodiimide (EDC) crosslinking method. The specific steps are as follows: Weigh 5 mg of KLH and dissolve it in 2 mL of 0.1 M MES buffer (pH 6.0). Add 3 mg of synthetic peptide GTTPSPVP and 10 mg of EDC, and react at room temperature in the dark for 2 hours; The reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed against PBS buffer (pH 7.4) at 4°C for 24 hours, with the dialysate being changed every 8 hours. The concentration of the conjugate was determined by the BCA method, and the product was aliquoted and stored at -80°C.

[0048] Animal immunization and spleen cell preparation: 6-8 week old female BALB / c mice were selected. The first immunization was performed using Freund's complete adjuvant emulsified immunogen (containing 50 μg peptide-KLH conjugate) and injected subcutaneously at multiple sites. Three booster immunizations were administered two weeks later, using Freund's incomplete adjuvant to emulsify the immunogen; Three days after the last immunization, the spleen of the mice was ground to obtain a spleen cell suspension, which was then treated with erythrocyte lysis buffer and washed three times with PBS.

[0049] Cell fusion and hybridoma screening: Spleen cells and SP2 / 0 myeloma cells were mixed at a ratio of 5:1 and fused for 90 seconds in 50% PEG-1500. The fused cells were resuspended in HAT medium (containing 20% ​​FBS and 1×HAT supplement) and seeded into 96-well plates and cultured in a 37°C, 5% CO2 incubator. After 10 days of culture, the medium was replaced with HT medium, and cultured until clones formed. Positive clones were screened using indirect ELISA: A GTTPSPVP-BSA coated plate (1 μg / well) was used as the solid-phase antigen, and hybridoma supernatant was added for incubation. HRP-labeled goat anti-mouse IgG secondary antibody was then used for color development, and OD... 450 > 1.0 is considered positive.

[0050] Cloning and antibody production involved three subclonings of positive hybridomas using a limiting dilution method until monoclonal cells were formed. Monoclonal hybridoma cells were amplified and injected into the peritoneal cavity of mice pretreated with phytane to produce ascites. Ascites antibodies were purified by Protein G affinity chromatography, and purity was verified by SDS-PAGE (>95%).

[0051] Antibody characterization and epitope specificity verification were performed, and threonine (T³) and proline (P⁻) in GTTPSPVP were confirmed by alanine scanning mutation (Ala-scan). 6 ) is a key binding residue (binding activity decreases by >90% after mutation); CDR3 functional verification: R of heavy chain CDR3 (CASRGPYFDYW) 5 The A mutation caused the KD value to increase from 0.5 nM to >1000 nM.

[0052] The APTES-glutaraldehyde crosslinking process for immobilizing lectin MAL-II on glass slides is as follows: Pre-treatment of glass slides: Immerse the glass slides in Piranha solution (concentrated sulfuric acid: 30% hydrogen peroxide = 3:1 v / v) and ultrasonically clean for 30 minutes; rinse with ultrapure water, blow dry with nitrogen, and place in an oven at 110℃ to dry for 1 hour.

[0053] APTES silanization treatment involves immersing a dry glass slide in an anhydrous ethanol solution containing 2% (v / v) 3-aminopropyltriethoxysilane (APTES) and reacting at 65°C for 1 hour. The slide is then ultrasonically cleaned with anhydrous ethanol and ultrapure water for 5 minutes in sequence, dried with nitrogen, and cured at 110°C for 30 minutes to form an aminated surface.

[0054] Aminated slides were activated by glutaraldehyde by immersing them in a 0.25% (v / v) glutaraldehyde solution in PBS (pH 7.4) and reacting at room temperature in the dark for 2 hours. The slides were then rinsed three times with ultrapure water and dried with nitrogen to obtain aldehyde-activated slides.

[0055] Lectin immobilization: MAL-II lectin (2 mg / mL dissolved in 0.1 M carbonate buffer, pH 9.0) was spotted onto the surface of an aldehyde-based glass slide (spotting volume: 0.5 μL / spot, spot spacing 200 μm). Place in a humidified chamber and react at 37°C for 3 hours to allow the primary amino group of the lectin to form a Schiff base with the aldehyde group. Unreacted aldehyde groups were blocked by immersion in 0.1 M glycine-PBS solution (pH 7.4) for 30 minutes. Wash three times with PBS containing 0.05% Tween-20, dry with nitrogen, and store at 4°C for later use.

[0056] Table 6. Verification of the immobilization effect of lectin MAL-II

[0057] Test Project method Qualification Standard Binding activity Cy5-labeled sialic acid glycan binding Fluorescence intensity ≥ 3 times that of the control slide stability Retest after storing at 4℃ for 30 days Activity retention ≥90% Nonspecific adsorption BSA solution (1 mg / mL) incubation Fluorescence signal ≤ 110% of negative control

[0058] This invention provides a method for detecting Riemerella anatipestifer infection, comprising the following steps: S1, Sample collection: Obtain duck feces, respiratory lavage fluid, or mucosal scrapings as samples; S2, use pretreatment solution to process the sample; S3: For fecal samples, use step S31; for respiratory lavage fluid samples, use step S33 or S31; for mucosal scraping samples, use step S32; Perform biomarker detection: S31, Detection and quantification of mucin cleavage fragments: S311, add the pretreated sample to an ELISA plate coated with anti-Muc5ac monoclonal antibody and incubate; S312, incubated with biotin-labeled anti-GTTPSPVP monoclonal antibody; S313, with the addition of streptavidin-HRP conjugate and TMB substrate for color development; S314, Quantitatively calculate the concentration of mucin cleavage fragments based on a standard curve, wherein the standard curve is established using a gradient dilution of recombinant protein containing the GTTPSPVP epitope; S32, Detection of sialylated glycan chains: S321, the pretreated sample is dropped onto the glass slide chip with immobilized MAL-II lectin; S322, incubated with Cy5 fluorescently labeled streptavidin; S323, scan fluorescence signals and calculate lectin binding values; Detection and concentration quantification of S33 and Muc2 degradation peptides: S331, Load the pretreated sample into the sample application area of ​​the immunochromatographic test strip; S332, through chromatography, the peptide shown in SEQ ID NO:1 in the sample binds to the coated anti-Muc2 polyclonal antibody; S333, based on the ratio of the color intensity of the test line to the control line on the test strip, the concentration of Muc2 degradation peptides is quantified with reference to the colorimetric card; S334, validation of the validity of the sheep anti-rabbit IgG quality control line results; S4. A positive result for Riemerella anatipestifer infection is determined when the test value meets any of the following conditions: Concentration of mucin cleavage fragments > 15 ng / mL; Sialized glycan binding value > 3 times that of negative control; The concentration of Muc2-degraded peptides is > 8 ng / mL.

[0059] The specific process for sample collection S1 involves operators obtaining duck fecal samples, respiratory lavage fluid samples, or respiratory / intestinal mucosal scraping samples as testing materials. Fecal samples facilitate large-scale non-invasive screening on farms; respiratory lavage fluid effectively enriches respiratory infection-related markers; and mucosal scrapings directly obtain mucosal tissue from the infected site, suitable for precise laboratory analysis. This multi-source sample design ensures the method can adapt to different testing scenarios and sample acquisition conditions.

[0060] The specific procedure for sample pretreatment S2: Operators must use a dedicated sample pretreatment solution to treat the collected samples. This pretreatment solution contains 50 mM Tris-HCl buffer (pH 8.0), 1% v / v Triton X-100, 5 mM EDTA, and a 10 μg / mL mixture of protease inhibitors.

[0061] Tris-HCl buffer maintains the reaction system within the optimal pH range for proteases; Triton X-100, as a surfactant, cleaves mucosal cell membranes, fully releasing intracellular and membrane-bound target biomarkers; EDTA effectively inhibits the activity of endogenous metalloproteinases in the sample by chelating metal ions such as Ca²⁺ and Zn²⁺, preventing their degradation of mucin fragments and Muc2 degradation peptides; the protease inhibitor mixture, containing AEBSF, E-64, etc., broadly inhibits serine proteases, cysteine ​​proteases, etc., maximizing the protection of the structural integrity of all three types of biomarkers. This pretreatment protocol is uniformly applicable to three sample types, simplifying the operation process and significantly improving biomarker recovery and stability.

[0062] The specific process for biomarker detection S3 is as follows: First, module selection is performed. For fecal samples, S31 mucin cleavage fragment detection is preferred. For respiratory lavage fluid samples, either S31 mucin cleavage fragment detection or S33 Muc2 degradation peptide detection can be used. For mucosal scraping samples, S32 sialylated glycan detection is selected.

[0063] The detection process is as follows: S31: Detection and quantification of mucin cleavage fragments, i.e., ELISA method; S311 Capture: The operator adds the pretreated sample to the wells of an ELISA plate pre-coated with anti-Muc5ac monoclonal antibody and incubates at 37°C for 60 minutes. The capture antibody anti-Muc5ac can specifically bind to the cleavage fragments produced by the virulence enzyme of Riemerella anatipestifer cleavage of the host Muc5ac protein in the sample, recognizing its complete protein backbone structure.

[0064] S312 Detection: After incubation, unbound material was washed away, and biotin-labeled anti-GTTPSPVP monoclonal antibody was added. The mixture was then incubated again at 37°C for 45 minutes. This detection antibody specifically recognizes the linear epitope GTTPSPVP SEQID NO:2 exposed by bacterial cleavage in the lysate fragment. The antibody's high affinity stems from the antigen-binding pocket formed by its heavy chain CDR3CASRGPYFDYW and light chain CDR3CQQYRTYPHVF. Key residues such as arginine R, tyrosine Y, and histidine H precisely lock onto the epitope amino acids, such as threonine T and proline P, through hydrogen bonding and hydrophobic interactions, constructing a double-antibody sandwich complex of anti-Muc5ac antibody-lysate fragment-anti-GTTPSPVP antibody.

[0065] S313 Signal Amplification and Color Development: After washing again, the operator added streptavidin-HRP horseradish peroxidase conjugate and incubated at room temperature in the dark for 30 minutes. Streptavidin binds with high affinity to the biotin-labeled detection antibody. Subsequently, a TMB 3,3',5,5'-tetramethylbenzidine substrate solution was added for incubation and color development. HRP catalyzes TMB to produce a blue soluble product, and the color intensity is proportional to the amount of target cleavage fragment bound.

[0066] S314 Quantification: The operator adds stop solution to terminate the reaction and reads the absorbance value at 450 nm wavelength on a microplate reader. The concentration of mucin cleavage fragments in the sample (ng / mL) can be quantified by comparing with a standard curve. The standard curve is established using serially diluted recombinant protein containing the GTTPSPVP epitope, followed by the same ELISA procedure. This method has high sensitivity, low detection limit, and accurate quantification, making it particularly suitable for detecting early infections and high-load samples.

[0067] S32: Detection of lectin-based sialized glycans using a lectin-based chip method; S321 Binding: The operator drops the pretreated sample, especially mucosal scrapings, onto a glass slide chip immobilized with lectin MAL-II and incubates it in a humidified chamber at 37°C for 60 minutes. MAL-II lectin (Maackiaamurensislectin II) exhibits high specificity and high affinity, recognizing and firmly binding to the terminal α2,6-linked sialyl-modified N-acetylgalactosamine motif Neu5Acα2-6GalNAcβ1-3Gal in biomarkers. This glycan structure is abnormally generated by the infection-activated host sialyltransferase ST6GalNAc1 and is competitively inhibited by α2,3-sialylation in healthy tissues.

[0068] S322 Signal Labeling: After incubation and washing, the operator adds a Cy5 anthocyanin 5 fluorescently labeled streptavidin solution and incubates at room temperature in the dark for 30 minutes. High-sensitivity fluorescent labeling is achieved through its binding with biotin, pre-biotinylation of the fructose chain, or signal amplification using a biotin-avidin system in chip design, or direct / indirect binding to Cy5.

[0069] S323 Signal Reading: The operator uses a laser scanner to scan the slide chip and detect the Cy5 fluorescence signal. The fluorescence intensity and lectin binding value of each point are calculated using analysis software. This method's lectin recognition of sugar structures is unaffected by bacterial antigen variation, exhibiting high specificity. Furthermore, the binding of MAL-II to α2,6-sialic acid is irreversible, resulting in stable signals. It is highly resistant to mucin interference in samples, making it particularly suitable for analyzing high-mucus samples such as respiratory lavage fluid.

[0070] S33: Muc2 Degradation Peptide Detection and Concentration Quantification Immunochromatographic Test Strip Method; S331 Sample Addition Chromatography: The operator adds the pretreated sample, especially suitable for rapid on-site detection of respiratory lavage fluid, to the sample addition area of ​​the immunochromatographic test strip. The sample is chromatographically deposited along the nitrocellulose membrane (NC membrane) of the test strip by capillary action.

[0071] S332 Capture: When the sample is chromatographyd to the detection line (T line), the Muc2 C-terminus-specific degradation peptide sequence (PRGDTVVVPC, SEQ ID NO: 1) in the sample is captured by an anti-Muc2 polyclonal antibody pre-coated on an NC membrane, forming an antibody-antigen complex. This antibody strictly recognizes the RGDArg-Gly-Asp integrin-binding domain and the hydrophobic core Val / Pro enriched region in the peptide.

[0072] S333 Signal Generation and Semi-Quantification: If the sample contains the target peptide, chromogenic nanoparticles will aggregate on the detection line, causing the T line to develop color. Simultaneously, regardless of the presence of the target peptide, the chromatography solvent continues to flow to the control line (C line), where it is captured by the coated goat anti-rabbit IgG, ensuring reagent effectiveness and chromatography completion. Operators can semi-quantify the concentration (ng / mL) of the Muc2-degraded peptide in the sample by referring to the color intensity ratio of the T and C lines and the concentration ranges corresponding to different intensity ratios provided by a standard colorimetric card. The operation is extremely simple, requiring only sample addition; the detection is rapid, typically completed within 10 minutes, requiring no special instruments, making it ideal for point-of-care testing (POCT) in livestock farms.

[0073] S334 Quality Control: The control line C, representing goat anti-rabbit IgG capture-labeled antibody, is used to verify the effectiveness of the chromatography process and reagent activity. Only when the C line shows normal color development is the test result considered valid. The NC membrane of the test strip is usually blocked with 0.1% BSA + 5% sucrose, which effectively reduces the non-specific adsorption of mucus components in the sample and improves accuracy.

[0074] The specific process for interpreting the results of S4 is as follows: the operator interprets the results based on the selected detection module. A positive result for *Rimeria anatidae* infection can be determined when any of the following conditions are met: mucin cleavage fragment concentration >15 ng / mL; sialylated glycohemagglutinin binding value >3 times the negative control value; or Muc2 degradation peptide concentration >8 ng / mL.

[0075] The detection principle for mucin cleavage fragments with a concentration >15 ng / mL is based on the quantitative detection of cleavage fragments produced by the specific cleavage of host Muc5ac mucin by the virulence enzyme sialidase / metalloproteinase of Riemerella anatipestifer in a sample using a double-antibody sandwich ELISA. This fragment contains the exposed linear epitope GTTPSPVPSEQID NO:2, which is undetectable in healthy tissue due to glycosylation masking.

[0076] The threshold was set based on ROC curve analysis: Validated using clinical samples from an infected group (n=120) and a healthy group (n=100), when 15 ng / mL was used as the cutoff value: Sensitivity: 98.3% positive detection rate in the infected group; Specificity: 96.0% negative concordance rate in the healthy group; Area under the AUC curve: 0.992, close to 1.0, indicating high discrimination. The biological basis is that the concentration of this fragment in the mucosa / feces of healthy ducks is generally <5 ng / mL (background noise level); the concentration reaches 8-12 ng / mL within 24 hours in the early stage of infection, and stabilizes at >20 ng / mL after 48 hours in the progression stage.

[0077] Enzymatic digestion can be detected even when the bacterial proliferation level is <10³ CFU / mL, breaking the PCR detection limit; only bacterial virulence enzymes can expose the GTTPSPVP epitope, and the concentration in common inflammations such as E. coli infection is <10 ng / mL.

[0078] The detection principle of the sialylated glycan binding value >3 times that of the negative control is as follows: the immobilized lectin MAL-II chip is used to capture the abnormally expressed sialylated glycan structure in the sample, with the Neu5Ac-GalNAcβ1,3Gal motif linked at the end α2,6-, and the fluorescence signal is quantitatively labeled by Cy5-streptavidin.

[0079] The threshold is set based on the following: lectin binding value = sample fluorescence intensity - blank control / negative control fluorescence intensity, and a value three times that of the sample is considered positive. The binding value of healthy mucosal samples is 0.8-1.2 times, because they are not recognized by MAL-II due to background α2,3-sialylation; the binding value of infected samples is 4.2-15.3 times, because bacteria activate the host ST6GalNAc1 enzyme and specifically generate α2,6 bonds; the binding value of samples infected by other pathogens such as duck plague virus is <1.5 times.

[0080] α2,6-sialic acid modification is resistant to sialylase degradation and exhibits stable signaling; it is unaffected by bacterial antigen variation in targeting host response products; MAL-II has a KD ≤ 10⁻ for α2,6-Sia. 8 M can detect pmol-level sugar chains.

[0081] The detection principle of the Muc2 degradation peptide concentration >8ng / mL LLS33 module is as follows: the immunochromatographic test strip quantitatively detects the specific peptide PRGDTVVVPC (SEQ ID NO:1) generated by the bacterial serine protease RapA cleaving the carboxyl terminus of Muc2 protein, and the quantitative determination is based on the color intensity ratio of the T / C line to the reference colorimetric card.

[0082] Table 7 Concentration gradient validation

[0083] Concentration (ng / mL) Color rendering intensity ratio (T / C) Result determination ≤2 <0.1 Negative 5 0.3 gray area **≥8** **≥0.5** Positive

[0084] The concentration of respiratory lavage fluid in infected ducks was 12-35 ng / mL (still >8 ng / mL during the recovery period, half-life 72h); in healthy / other diseased samples, 92% had concentrations <5 ng / mL (false positive rate <8%). Rapid on-site testing yields results in 10 minutes, requiring no instruments (visual interpretation via color chart); recovery period monitoring utilizes the slow metabolism of short peptides to assess treatment efficacy; anti-matrix interference is achieved by blocking the test strip with 0.1% BSA + 5% sucrose, resulting in a false positive rate of <5% for mucus samples.

[0085] The three biomarkers correspond to different infection stages and sites: mucin fragments are used for early intestinal / respiratory infections (highest sensitivity); sialylated glycans are used for mucosal colonization in the progressive stage (strong resistance to mutation); and the Muc2 peptide is used for monitoring respiratory-specific damage and the recovery period (most convenient operation). Combined use can cover the entire disease course (detection rate >99.5%), while a single module meets POCT needs. Threshold setting was optimized using receiver operating characteristic (ROC) curves to maximize sensitivity and specificity. The experimental examples and results of clinical application are as follows: Experimental Case 1 (Early Infection): Ducks showed respiratory symptoms 12 hours after infection, and PCR tests were negative.

[0086] Using the S31 step: a mucin fragment concentration of 18 ng / mL (>15 ng / mL) is considered positive, earlier than pathogen detection.

[0087] Experimental Example 2 (Difficult Sample): Excessive mucus in the mucosal scraping results in a high ELISA background.

[0088] Using step S32: The lectin binding value is 4.8 times > 3 times, which is considered positive, indicating that the mucus interference has been overcome.

[0089] Experimental Example 3 (On-site Screening): The farm was quickly inspected, but lacked specialized equipment.

[0090] Using the S33 procedure: Test strip T / C=0.7, corresponding to ≈12 ng / mL >8 ng / mL, is considered positive, and confirmation is achieved in 10 minutes.

[0091] The positive threshold, determined based on extensive experimental data, aims to balance sensitivity and specificity. This interpretation criterion allows for flexible selection of a single module for detection, making it particularly suitable for rapid on-site testing, and also supports the combined application of multiple modules. Multi-marker combined detection strategies can significantly improve diagnostic reliability, avoid the false negative or false positive risks of single methods, and achieve effective coverage of different stages of infection (early enzymatic cleavage, progressive glycosylation, and residual peptides in the recovery phase). The positive threshold is based on host-pathogen interaction-specific molecular events, including enzymatic epitope exposure, rare glycosylation modification, and protease-cleaved peptides, and is a decision boundary established through rigorous biostatistical validation. It differs from the non-specific elevations seen in common inflammation; it balances precise laboratory quantification with rapid on-site screening; it precedes pathogen detection; it is resistant to sample interference; and it tolerates antigenic variations.

[0092] This invention employs three biomarkers: enzyme-exposed mucin epitopes, abnormally expressed α2,6-sialylated sugar chains, and Muc2 peptides produced by bacterial protease cleavage. These are all produced by the host induced by specific virulence factors (sialic acidase, metalloproteinase, and serine protease) of Riemerella anatipestifer, and are non-universal inflammatory response products, thus ensuring the specificity of the detection.

[0093] It offers three detection technology platforms (highly sensitive ELISA, highly interference-resistant lectin chip, and fast and convenient chromatography test strips), optimized for different sample types (feces, irrigation fluid, scrapings) and detection needs (precise quantification in the laboratory, rapid screening on-site), covering all application scenarios.

[0094] The carefully designed pretreatment solutions (chelating agents, protease inhibitors, detergents) effectively protect the biomarkers; each detection module employs highly specific recognition elements (customized monoclonal antibodies against CDR3, high-affinity lectin MAL-II, and polyclonal antibodies against short peptides) and optimized conditions (blocking buffer, specific pH coating buffer) to minimize non-specific binding and background interference.

[0095] The use of mature biotin-streptavidin systems (ELISA, chip) or colorimetric / fluorescent labeling (chromatography, chip) for signal amplification improves sensitivity; the irreversibility or stability of the target biomarkers (enzyme epitopes, rare sugar modifications, cleaved peptides) ensures the detection window period.

[0096] Based on a clearly defined positive threshold from experimental data, combined with the built-in quality control strip C-line, standard curve, and negative control, the accuracy and reproducibility of result interpretation are ensured. The multi-biomarker combined strategy further enhances diagnostic confidence.

[0097] Mucin cleavage markers can appear in the early stages of infection, earlier than bacterial proliferation, breaking through the detection bottleneck of traditional methods such as PCR or bacterial culture at low bacterial counts; immunochromatographic test strips meet the urgent need for rapid on-site diagnosis.

[0098] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A biomarker for detecting Riemerella anatipestifer infection, characterized in that: The biomarker is a host mucosal damage product induced by specific virulence factors of Riemerella anatipestifer after duck infection, and is selected from any of the following: Characteristic mucin cleavage fragments shed from intestinal or respiratory mucosal cells, with an isoelectric point pI of 4.5–5.2, are produced by cleavage of host Muc5ac protein by sialidase or metalloproteinase of Riemerella anatipestifer, and their sequence SEQ ID NO:2 contains the linear epitope GTTPSPVP. The aberrantly expressed sialylated glycan structure contains a terminal α2,6-linked sialic acid-modified N-acetylgalactosamine GalNAc-β1,3-Gal motif; The sequence of the carboxyl-terminal degradation peptide of the Muc2 protein, specifically secreted by goblet cells in the respiratory tract, is shown in SEQ ID NO:

1.

2. A kit for detecting Riemerella anatipestifer infection, characterized in that, Includes sample pretreatment solution, TMB substrate solution, signal intensity colorimetric card, and: The mucin cleavage fragment detection module includes an ELISA plate coated with anti-Muc5ac monoclonal antibody and biotin-labeled anti-GTTPSPVP monoclonal antibody; The sialylated glycan detection module includes a glass slide chip with immobilized lectin MAL-II and Cy5 fluorescently labeled streptavidin; The Muc2 degradation peptide detection module includes an immunochromatographic test strip containing anti-Muc2 polyclonal antibody and goat anti-rabbit IgG coated on a nitrocellulose membrane.

3. The kit according to claim 2, characterized in that: The heavy chain CDR3 sequence of the anti-GTTPSPVP monoclonal antibody is CASRGPYFDYW (SEQ ID NO:3), and the light chain CDR3 sequence is CQQYRTYPHVF (SEQ ID NO:4); the enzyme-labeled plate coating buffer is carbonate buffer at pH 9.

6.

4. The kit according to claim 3, characterized in that, In the sialylated glycan detection module, immobilized lectin MAL-II specifically binds to the sialylic acid-modified N-acetylgalactosamine GalNAc-β1,3-Gal motif linked at the terminal α2,6-; Cy5 fluorescently labeled streptavidin detects the biotin-labeled lectin-glycan complex.

5. The kit according to claim 4, characterized in that, In the Muc2 degradation peptide detection module, the anti-Muc2 polyclonal antibody specifically recognizes the peptide sequence of SEQ ID NO:1; the goat anti-rabbit IgG is the quality control line of the test strip.

6. The reagent kit as described in claim 5, characterized in that, The sample pretreatment solution contained 50 mM Tris-HCl buffer, 1% Triton X-100, 5 mM EDTA, and a 10 μg / mL mixture of protease inhibitors.

7. A method for detecting Riemerella anatipestifer infection using the kit as described in claim 5, characterized in that, Includes the following steps: S1, Sample collection: Obtain duck feces, respiratory lavage fluid, or mucosal scrapings as samples; S2, Use pretreatment solution to treat the sample; S3: For fecal samples, use step S31; for respiratory lavage fluid samples, use step S33 or S31; for mucosal scraping samples, use step S32; Perform biomarker detection: S31, Detection and quantification of mucin cleavage fragments: S311, add the pretreated sample to an ELISA plate coated with anti-Muc5ac monoclonal antibody and incubate; S312, incubated with biotin-labeled anti-GTTPSPVP monoclonal antibody; S313, with the addition of streptavidin-HRP conjugate and TMB substrate for color development; S314, Quantitatively calculate the concentration of mucin cleavage fragments based on a standard curve, wherein the standard curve is established using a gradient dilution of recombinant protein containing the GTTPSPVP epitope; S32, Detection of sialylated glycan chains: S321, the pretreated sample is dropped onto the glass slide chip with immobilized MAL-II lectin; S322, incubated with Cy5 fluorescently labeled streptavidin; S323, scan fluorescence signals and calculate lectin binding values; Detection and concentration quantification of S33 and Muc2 degradation peptides: S331, Load the pretreated sample into the sample application area of ​​the immunochromatographic test strip; S332, through chromatography, the peptide shown in SEQ ID NO:1 in the sample binds to the coated anti-Muc2 polyclonal antibody; S333, based on the ratio of the color intensity of the test line to the control line on the test strip, the concentration of Muc2 degradation peptides is quantified with reference to the colorimetric card; S334, validation of the validity of the sheep anti-rabbit IgG quality control line results; S4. A positive result for Riemerella anatipestifer infection is determined when the test value meets any of the following conditions: Concentration of mucin cleavage fragments > 15 ng / mL; Sialized glycan binding value > 3 times that of negative control; The concentration of Muc2-degraded peptides is > 8 ng / mL.

Images