Elisa kit for distinguishing listeria monocytogenes and / or listeria ivanovii infection, and preparation method therefor and use thereof

Abstract

An indirect ELISA detection kit for distinguishing Listeria monocytogenes and / or a Listeria ivanovii infection in a host. Coated antigens are respectively recombinant Listeria monocytogenes surface protein ActA, common outer-membrane protein SmcL of Listeria monocytogenes serotype 4h and Listeria ivanovii, and Listeria ivanovii-specific outer-membrane protein I-inlH. Indirect ELISA methods are established for the three antigens, respectively. By means of combining the three detection methods, the levels of the ActA antibody, SmcL antibody and I-inlH antibody in a clinical serum sample can be effectively monitored, and the detection of listeriosis caused by Listeria monocytogenes and / or Listeria ivanovii is realized.

Description

ELISA kit for distinguishing Listeria monocytogenes and / or Listeria ivanovii infection, preparation method and application thereof Technical Field

[0001] The present invention relates to the technical field of antibody detection, and in particular to an indirect ELISA kit for detecting antibodies against pathogenic Listeria in serum. Background Art

[0002] Listeria monocytogenes (Lm) and Listeria ivanovii (Liv) are the main pathogenic species of the genus Listeria, causing listeriosis in both humans and animals. L. monocytogenes infection of animals can cause severe economic losses to livestock farms. L. monocytogenes carried by animals enters the food chain through slaughter and processing, and then continues through the processing, transportation, and sale of animal products to the human table, where it can cause human infection, with a mortality rate as high as 20%-30%. Most listeriosis cases are caused by strains of serotypes 1 / 2b and 4b of lineage I and serotype 1 / 2a of lineage II. The virulent strain ST626 isolated from an outbreak of listeriosis on a sheep farm belongs to the newly designated serotype 4h of L. monocytogenes. L. ivanovii primarily infects ruminants, and sheep are a susceptible reservoir for Listeria in nature. Sheep listeriosis causes significant economic losses to the livestock industry. In recent years, there have been reports of cases of human gastroenteritis and sepsis caused by Listeria ivanovii, which require high attention.

[0003] Strengthening listeriosis surveillance in the livestock sector will enable the detection of infected animals on farms and prompt action to prevent diseased livestock from entering the food supply chain, which is of great significance to public health and safety. The gold standard for listeriosis diagnosis is the isolation of the pathogen. However, this method is time-consuming and often leads to inaccurate test results due to factors such as the short lifespan of viable bacteria and irregular bacterial excretion. Serological tests are commonly used in the auxiliary diagnosis of infectious diseases, complementing and supporting traditional bacterial isolation and culture methods. Among them, the ELISA method has the advantages of ease of operation, rapid speed, and the ability to screen large quantities of samples at high throughput, making it widely used in epidemiological surveys of sheep farms and clinical immune monitoring. Currently, there is a commercially available diagnostic kit for listeriosis, an indirect ELISA based on listerolysin O (LLO). This kit cannot distinguish between infections caused by Listeria monocytogenes and Listeria ivanovii, and cross-reacts with serum antibodies following infection with other bacterial genera such as Streptococcus.

[0004] ActA is a key virulence factor of Listeria monocytogenes, located on the cell wall and proven to be highly immunogenic. SmcL is an outer membrane protein shared by both 4h-type Listeria monocytogenes and Listeria ivanovii. I-inlH is a unique outer membrane protein of Listeria ivanovii.

[0005] There is an urgent need in the prior art for a test kit that can simultaneously and distinguish between detecting Listeria monocytogenes and Listeria ivanovii.

[0006] Summary of the Invention

[0007] To address the deficiencies in the prior art, the present invention selects the Listeria monocytogenes surface protein ActA, the outer membrane protein SmcL shared by serotype 4h Listeria monocytogenes and Listeria ivanovii, and the outer membrane protein I-inlH of Listeria ivanovii as detection antigens, establishes an indirect ELISA detection method, and prepares a kit. These methods can not only detect whether a sample is infected with Listeria, but also distinguish whether a sample is infected with Listeria monocytogenes and / or Listeria ivanovii, and further distinguish which type of Listeria monocytogenes is infected, whether it is infection with serotype 4h Listeria monocytogenes or infection caused by other Listeria monocytogenes.

[0008] The present invention provides an indirect ELISA kit for detecting Listeria monocytogenes and / or Listeria ivanovii, characterized in that the kit comprises carriers coated with ActA, SmcL and I-inlH proteins, respectively, a standard positive control and a standard negative control, and a detection reagent for detecting antigen-antibody reactions between ActA, SmcL and I-inlH proteins and antibodies thereto.

[0009] The present invention, for the first time, uses the combined use of ActA, SmcL, and I-inlH antigens to detect host infection with Listeria monocytogenes and / or Listeria ivanovii with high specificity and sensitivity. Furthermore, the inventors discovered for the first time that these three proteins exhibit good conservation and antigenicity, enabling their use as coating antigens in the preparation of indirect ELISA kits. In light of this, the inventors have developed and designed an indirect ELISA kit for distinguishing between infections with Listeria monocytogenes and Listeria ivanovii species.

[0010] The present invention also provides a method for preparing the indirect ELISA kit, characterized in that the method comprises the following steps: (1) coating: coating ActA, SmcL and I-inlH proteins on an enzyme-labeled plate respectively, wherein the amino acid sequence of the ActA protein is shown in SEQ ID NO.1, the amino acid sequence of the SmcL protein is shown in SEQ ID NO.2, and the amino acid sequence of the I-inlH protein is shown in SEQ ID NO.3; (2) blocking: blocking the enzyme-labeled plate coated with ActA, SmcL and I-inlH proteins in step (1) using a blocking solution; (3) preparing a standard positive control and a standard negative control; (4) preparing an enzyme-labeled secondary antibody and a color developing solution; assembling the enzyme-labeled plate prepared in step (2), the standard positive control and the standard negative control prepared in step (3), the enzyme-labeled secondary antibody prepared in step (4), and the color developing solution into the indirect ELISA kit.

[0011] The present invention optimizes the conditions for preparing an indirect ELISA kit, thereby preparing an indirect ELISA kit for distinguishing and detecting Listeria monocytogenes and Listeria ivanovii species with high specificity and sensitivity.

[0012] The present invention further provides a use of the indirect ELISA kit, which is used in the preparation of a detection reagent for detecting Listeria monocytogenes and / or Listeria ivanovii. The indirect ELISA kit of the present invention enables simultaneous and differentiated detection of Listeria monocytogenes and / or Listeria ivanovii, and can also differentiate between infection caused by serotype 4h Listeria monocytogenes and infection caused by other Listeria monocytogenes.

[0013] As described above, the indirect ELISA kit, preparation method, and application for detecting Listeria monocytogenes and / or Listeria ivanovii have the following beneficial effects:

[0014] 1) This study cloned and expressed the recombinant Listeria ivanovii protein I-inlH for the first time. Immunoblotting revealed that the recombinant ActA, SmcL, and I-inlH proteins all exhibited good reactogenicity. Furthermore, infection of sheep with 4h-type Listeria monocytogenes strain XYSN induced the production of specific antibodies against ActA and SmcL. Furthermore, infection of mice with Listeria ivanovii strain XYSL produced specific antibodies against I-inlH, demonstrating that ActA, SmcL, and I-inlH all exhibited good immunogenicity.

[0015] 2) The present invention's method and kit for detecting antibodies to Listeria monocytogenes and / or Listeria ivanovii can diagnose whether a host has listeriosis and can also determine the species of Listeria that infects the host. The kit can distinguish between L. monocytogenes and / or L. ivanovii infections in hosts with high sensitivity, specificity, and stability. The present invention minimizes false positive rates and avoids interference from antigens of other Listeria bacteria. Compared to the indirect ELISA method based on LLO antigens, which is currently widely used in veterinary clinics, the present invention has higher specificity.

[0016] 3) The establishment of this method fills the gap in serological detection methods for distinguishing Listeria monocytogenes and Listeria ivanovii infections, and can be used for the investigation of Listeria prevalence in host fields and clinical immune monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG1 shows an exemplary embodiment of the present invention and, together with the description, serves to explain the principles of the present invention. These drawings are included to provide a further understanding of the present invention and are incorporated in and constitute a part of this specification.

[0018] Figure 1A, B, and C are schematic diagrams of PCR amplification of the actA, smcL, and i-inlH genes, respectively; wherein, lane M: DL2000 DNA molecular mass standard; lanes 1 and 2: actA, smcL, and i-inlH gene amplification products, respectively;

[0019] Figures 2A, B, and C are schematic diagrams of double enzyme digestion identification of recombinant plasmids inserted with the actA, smcL, and i-inlH genes, respectively; wherein, lane M: DL5000 DNA molecular mass standard; lane 1: double enzyme digestion products of recombinant plasmids pET-30a-actA, pET-30a-smcL, and pET-30a-i-inlH; lane 2: undigested recombinant plasmids pET-30a-actA, pET-30a-smcL, and pET-30a-i-inlH;

[0020] Figures 3A, B, and C are schematic diagrams of induced expression in BL21(DE3)-pET-30a actA, BL21(DE3)-pET-30a smcL, and BL21(DE3)-pET-30a i-inlH transformants, respectively; wherein, lane M: low molecular weight protein quality standard; lane 1: purified rHis-ActA / rHis-SmcL / rHis-I-inlH fusion protein;

[0021] Figures 4A, B, and C are schematic diagrams of Western blot analysis of recombinant ActA, SmcL, and I-inlH proteins, respectively; wherein, lane M: low molecular weight protein quality standard; lane 1: purified recombinant rHis-ActA / rHis-SmcL / rHis-I-inlH fusion protein;

[0022] Figures 5A, B, C, and D are schematic diagrams of in vitro bioreactivity assays for ActA, SmcL, and I-inlH antigens; wherein, lane M: low molecular weight protein quality standard; lane 1: rGST-ActA; lane 2: rHis-SmcL A and B, rGST-SmcLB and C; lane 3: rHis-I-inlHC and D, rGST-I-inlH A and B;

[0023] Figures 6A, B, and C are graphs showing the effects of different antigen coating concentrations and enzyme-labeled secondary antibody dilutions on the OD450 values ​​of positive serum in the indirect ELISA methods for ActA, SmcL, and I-inlH, respectively;

[0024] Figures 7A, B, and C are graphs showing the effects of different coating conditions on P / N values ​​in the indirect ELISA methods for ActA, SmcL, and I-inlH, respectively;

[0025] Figures 8A, B, and C are graphs showing the effects of different blocking solutions on P / N values ​​in the indirect ELISA methods for ActA, SmcL, and I-inlH, respectively;

[0026] Figures 9A, B, and C are graphs showing the effect of different blocking times on P / N values ​​in the indirect ELISA methods for ActA, SmcL, and I-inlH, respectively;

[0027] Figures 10A, B, and C are graphs showing the effect of different incubation times of serum primary antibodies on P / N values ​​in the ActA, SmcL, and I-inlH indirect ELISA methods, respectively;

[0028] Figures 11A, B, and C are graphs showing the effect of different enzyme-labeled secondary antibody reaction times on P / N values ​​in the indirect ELISA methods for ActA, SmcL, and I-inlH, respectively;

[0029] Figures 12A, B, and C are graphs showing the effect of different substrate development times on P / N values ​​in the indirect ELISA methods for ActA, SmcL, and I-inlH, respectively;

[0030] Figures 13A, B and C are schematic diagrams of the changes in serum IgG antibodies over time after 4h-type Listeria monocytogenes strain XYSN infected Hu sheep and Listeria ivanovii strain XYSL infected mice detected by indirect ELISA methods, respectively. DETAILED DESCRIPTION

[0031] The present invention is described in detail below:

[0032] The present invention provides an indirect ELISA kit for detecting Listeria monocytogenes and / or Listeria ivanovii, comprising a carrier coated with ActA, SmcL, and I-inlH proteins, respectively; a standard positive control; a standard negative control; and a detection reagent for detecting antigen-antibody reactions between ActA, SmcL, and I-inlH proteins and their antibodies. Listeria monocytogenes is a small, Gram-positive bacillus lacking spores or a capsule. It can be divided into 11 serotypes based on its somatic and flagellar antigens. It was isolated from naturally infected rabbits in 1926 and named L. monocytogenes due to the increase in monocytes in the infected animals. It is a major pathogen that causes disease in animals and humans.

[0033] Listeria ivanovii is another pathogenic Listeria species. It only causes disease in animals, mainly infecting ruminants. Sheep are susceptible hosts of Listeria in nature, and sheep listeriosis has caused huge economic losses to the breeding industry.

[0034] ActA protein is an important virulence factor of Listeria monocytogenes. It is located on the cell wall surface and has been shown to have strong immunogenicity.

[0035] SmcL protein is an outer membrane protein common to Listeria monocytogenes and Listeria ivanovii.

[0036] The I-inlH protein is an outer membrane protein unique to Listeria ivanovii.

[0037] The mechanism of the ELISA detection process of the present invention: The basis of ELISA is the solid phase of antigens or antibodies and the enzyme labeling of antigens or antibodies. The antigens or antibodies bound to the surface of the solid phase carrier still retain their immunological activity, and the enzyme-labeled antibodies retain both their immunological activity and enzyme activity. During the measurement of the present invention, the specimen to be tested (the antibody to be tested therein) reacts with the antigen on the surface of the solid phase carrier. The antigen-antibody complex formed on the solid phase carrier is separated from other substances in the liquid by washing. The enzyme-labeled antibody is then added and also bound to the solid phase carrier through reaction. At this time, the amount of enzyme on the solid phase is in a certain ratio to the amount of the test substance in the specimen. After the substrate for the enzyme reaction is added, the substrate is catalyzed by the enzyme to form a colored product. The amount of the product is directly related to the amount of the test substance in the specimen, so qualitative or quantitative analysis can be performed based on the depth of the color.

[0038] This study first confirmed that three proteins—ActA, SmcL, and I-inlH—are immunogenic and can be used to detect infections caused by these pathogens. Their combined use can simultaneously and differentially detect Listeria monocytogenes and / or Listeria ivanovii, and can also distinguish between infections caused by serotype 4h Listeria monocytogenes and other Listeria monocytogenes.

[0039] As certain embodiments of the present invention, the amino acid sequence of the ActA protein is shown as SEQ ID NO.1, the amino acid sequence of the SmcL protein is shown as SEQ ID NO.2, and the amino acid sequence of the I-inlH protein is shown as SEQ ID NO.3.

[0040] The amino acid sequence of the ActA protein has ≥90% homology with the sequence shown in SEQ ID NO.1; preferably, the amino acid sequence of the ActA protein has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology with the sequence shown in SEQ ID NO.1.

[0041] The amino acid sequence of the SmcL protein has ≥90% homology with the sequence shown in SEQ ID NO.2; preferably, the amino acid sequence of the SmcL protein has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology with the sequence shown in SEQ ID NO.2.

[0042] The amino acid sequence of the I-inlH protein has ≥90% homology with the sequence shown in SEQ ID NO.3; preferably, the amino acid sequence of the I-inlH protein has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology with the sequence shown in SEQ ID NO.3.

[0043] As one embodiment of the present invention, the coding gene sequences of ActA, SmcL and I-inlH recombinant proteins are shown in SEQ ID NOs. 4, 5 and 6.

[0044] As certain embodiments of the present invention, the antibodies are monoclonal antibodies or polyclonal antibodies against ActA protein, monoclonal antibodies or polyclonal antibodies against SmcL protein, and monoclonal antibodies or polyclonal antibodies against I-inlH protein; the detection reagents are secondary antibodies labeled with markers and substrate color development solution.

[0045] In certain embodiments of the present invention, the label is horseradish peroxidase (HRP), alkaline phosphatase, or biotin.

[0046] As certain embodiments of the present invention, the substrate color developing solution is 3,3',5,5'-tetramethylbenzidine (TMB).

[0047] As certain embodiments of the present invention, the indirect ELISA kit includes a sample diluent, a washing solution, a color developing solution and a stop solution.

[0048] As certain embodiments of the present invention, the sample diluent is a PBST solution containing 1% BSA, the washing solution is a PBST solution with pH 7.2 containing 0.05% Tween 20, and the stop solution is a 2 mol / L H2SO4 solution.

[0049] As certain embodiments of the present invention, the standard positive control comprises positive serum from a host infected with serotype 4h Listeria monocytogenes, other Listeria monocytogenes, or Listeria ivanovii, respectively.

[0050] As certain embodiments of the present invention, the standard negative control comprises negative serum from a host that is not infected with serotype 4h Listeria monocytogenes, other Listeria monocytogenes, or Listeria ivanovii.

[0051] As certain embodiments of the present invention, the carriers coated with ActA, SmcL and I-inlH proteins respectively include ELISA plates.

[0052] The present invention also provides a method for preparing the indirect ELISA kit, which comprises the following steps:

[0053] (1) Coating: Coating ActA, SmcL and I-inlH proteins on an ELISA plate, respectively. The amino acid sequence of the ActA protein is shown in SEQ ID NO.1, the amino acid sequence of the SmcL protein is shown in SEQ ID NO.2, and the amino acid sequence of the I-inlH protein is shown in SEQ ID NO.3. (2) Blocking: Blocking the ELISA plate coated with ActA, SmcL and I-inlH proteins in step (1) with a blocking solution. (3) Preparing a standard positive control and a standard negative control. Preferably, the standard positive control includes the standard negative control. (4) Preparing a secondary antibody labeled with a marker and a substrate color development solution. Assembling the ELISA plate prepared in step (2), the standard positive control and the standard negative control prepared in step (3), the labeled secondary antibody prepared in step (4), and the substrate color development solution into the indirect ELISA kit.

[0054] As certain embodiments of the present invention, the ActA, SmcL, and I-inlH proteins described in step (1) are respectively dissolved in CBS coating solution and coated on an ELISA plate at final concentrations of 0.625, 0.625, and 1.25 μg / ml, respectively. The ActA-coated plate is coated at 37°C for 2 hours, and the SmcL and I-inlH-coated plates are coated at 4°C for 16 hours. As certain embodiments of the present invention, the detection kit contains one ELISA plate each coated with ActA, SmcL, and I-inlH proteins.

[0055] As certain embodiments of the present invention, the coating antigen can be pre-coated on an ELISA plate, or a blank ELISA plate and a detection antigen can be provided, and the operator can coat the detection antigen on the ELISA plate using a prescribed method before testing.

[0056] As certain embodiments of the present invention, the antigen coating solution is a 0.05 mol / L carbonate (CBS) buffer solution at pH 9.6 containing ActA, SmcL or I-inlH protein, respectively.

[0057] As certain embodiments of the present invention, in step (2), the ActA-coated plate is blocked with PBS containing 5% BSA for 2.5 h, and the SmcL- and I-inlH-coated plates are blocked with PBS containing 5% skim milk for 1.5 h.

[0058] As certain embodiments of the present invention, the method further comprises preparing a sample diluent, a washing solution, a color developing solution, and a stop solution.

[0059] In certain embodiments of the present invention, the label is horseradish peroxidase (HRP), alkaline phosphatase, or biotin.

[0060] As certain embodiments of the present invention, the enzyme-labeled secondary antibody is a rabbit anti-goat IgG antibody labeled with horseradish peroxidase.

[0061] As certain embodiments of the present invention, the sample diluent is a PBST solution containing 1% w / v BSA.

[0062] As certain embodiments of the present invention, the washing solution is a PBST solution containing 0.05% w / v Tween 20 at pH 7.2.

[0063] As certain embodiments of the present invention, the color developing solution is 3,3',5,5'-tetramethylbenzidine (TMB).

[0064] As certain embodiments of the present invention, the stop solution is a 2 mol / L H2SO4 solution.

[0065] As certain embodiments of the present invention, the optimal coating concentration of ActA protein is 0.625 μg / mL, the coating conditions are 37° C. for 2 h, the blocking conditions are PBS solution containing 5% BSA for 2.5 h, and the working concentration of the enzyme-labeled secondary antibody is 1:40,000.

[0066] As certain embodiments of the present invention, the coating concentration of the SmcL protein is 0.625 μg / mL, the coating conditions are 16 h at 4°C, the blocking conditions are 1.5 h with a PBS solution containing 5% skim milk powder, and the working concentration of the enzyme-labeled secondary antibody is 1:10,000.

[0067] The present invention also provides an application of the indirect ELISA kit, which is an application of the indirect ELISA kit in preparing a detection reagent for detecting Listeria monocytogenes and / or Listeria ivanovii.

[0068] As certain embodiments of the present invention, the detection reagent for detecting Listeria monocytogenes and / or Listeria ivanovii is a detection reagent for distinguishing and detecting Listeria monocytogenes from Listeria ivanovii.

[0069] As certain embodiments of the present invention, the detection reagent for detecting Listeria monocytogenes and / or Listeria ivanovii is a detection reagent for distinguishing and detecting serotype 4h Listeria monocytogenes or other Listeria monocytogenes.

[0070] The present invention also provides a method for detecting Listeria monocytogenes and / or Listeria ivanovii using the indirect ELISA kit, the method comprising the following steps:

[0071] (1) Incubation of test samples: Dilute the test samples with diluent, incubate the diluted test samples on ActA-coated plates, SmcL-coated plates, and I-inlH-coated plates, and add the respective standard negative control and standard positive control;

[0072] (2) Secondary antibody incubation: adding secondary antibody to the ActA-coated plate, SmcL-coated plate, and I-inlH-coated plate to which serum was added in step (1), and incubating;

[0073] (3) Color development: Add the detection reagent to the coated plate incubated with the secondary antibody in step (2), develop color, then add the stop solution to stop the color development, and read the absorbance at OD450nm;

[0074] (4) Result determination: The ELISA method uses the S / P ratio for determination, and the formula is S / P = (sample OD450nm value - negative control OD450nm average) / (positive control OD450nm average - negative control OD450nm average); for ActA detection results, an S / P ratio greater than the critical value of 0.104 is judged as positive, otherwise it is judged as negative; for SmcL detection results, an S / P ratio greater than the critical value of 0.110 is judged as positive, otherwise it is judged as negative; for I-inlH detection results, an S / P ratio greater than the critical value of 0.137 is judged as positive, otherwise it is judged as negative. As certain embodiments of the present invention, the ActA, SmcL and I-inlH proteins are recombinant proteins, and their preparation method comprises the following steps:

[0075] (a) PCR amplification was performed using the genome of Listeria monocytogenes LM strain 4 as a template to clone a truncated actA gene fragment; PCR amplification was performed using the genome of Listeria ivanovii strain XYSL as a template to clone truncated smcL and i-inlH gene fragments;

[0076] (b) The nucleic acid fragments of the recombinant protein were separated by electrophoresis, recovered and purified by gel ligation with the Escherichia coli prokaryotic expression vector pET-30a to obtain recombinant plasmids pET-30a-actA, pET-30a-smcL and pET-30a-i-inlH; and transformed into DH5α competent cells to obtain prokaryotic recombinant strains; the plasmids were extracted and subjected to double enzyme digestion electrophoresis detection, and the recombinant plasmids identified as correct by electrophoresis were sequenced and analyzed;

[0077] (c) transforming the recombinant plasmid into Escherichia coli BL21 (DE3) competent cells for prokaryotic expression to obtain three recombinant proteins, namely, Listeria monocytogenes rHis-ActA protein and Listeria ivanovii rHis-SmcL and rHis-I-inlH proteins;

[0078] (d) The prokaryotic expression bacterial solution was sonicated and then centrifuged to obtain a supernatant containing the target protein. The supernatant was affinity chromatographed on a His fusion protein purification column, and the eluate after the column was collected to obtain purified ActA, SmcL, and I-inlH recombinant proteins.

[0079] As certain embodiments of the present invention, in step (a), the primer pair sequences used for amplification are shown as SEQ ID NOs. 7, 8, 9, 10, 11 and 12.

[0080] In a specific ELISA test of serum samples, the optimal coating concentration for ActA antigen was 0.625 μg / mL, the optimal coating condition was 2 hours at 37°C, the optimal blocking condition was 2.5 hours with 5% BSA in PBS, the optimal serum exposure time was 1 hour, the optimal enzyme-labeled secondary antibody working concentration was 1:40,000, the optimal enzyme-labeled secondary antibody exposure time was 30 minutes, and the optimal substrate development time was 5 minutes. Under these conditions, an S / P ratio of (SN) / (PN) greater than 0.104 was considered ActA positive, and vice versa.

[0081] In a specific ELISA test of serum samples, the optimal coating concentration for SmcL antigen was 0.625 μg / mL, the optimal coating condition was 16 hours at 4°C, the optimal blocking condition was 1.5 hours with 5% skim milk powder in PBS, the optimal serum exposure time was 2.5 hours, the optimal enzyme-labeled secondary antibody working concentration was 1:10,000, the optimal enzyme-labeled secondary antibody exposure time was 60 minutes, and the optimal substrate development time was 10 minutes. Under these conditions, a SmcL-positive result was determined when S / P = (SN) / (PN) > 0.110, and a negative result otherwise.

[0082] In a specific ELISA test of serum samples, the optimal coating concentration for I-inlH antigen was 1.25 μg / mL, the optimal coating condition was 16 hours at 4°C, the optimal blocking condition was 1.5 hours with 5% skim milk powder in PBS, the optimal serum exposure time was 2.5 hours, the optimal enzyme-labeled secondary antibody working concentration was 1:20,000, the optimal enzyme-labeled secondary antibody exposure time was 60 minutes, and the optimal substrate development time was 10 minutes. Under these conditions, an I-inlH positive result was determined when S / P = (SN) / (PN) > 0.137; otherwise, it was considered negative.

[0083] As certain embodiments of the present invention, the test samples in step (1) are incubated on ActA-coated plates at 37°C for 1 hour, and on SmcL- and I-inlH-coated plates at 37°C for 2.5 hours.

[0084] As certain embodiments of the present invention, the test sample in step (1) is serum.

[0085] As certain embodiments of the present invention, in step (2), 100 μl of a secondary antibody diluted 1:20,000 is added to the ActA-coated plate and the SmcL-coated plate of step (1) and incubated for 60 min; and 100 μl of a secondary antibody diluted 1:10,000 is added to the I-inlH-coated plate and incubated for 60 min.

[0086] As certain embodiments of the present invention, in step (3), the color development time of the ActA-coated plate is 5 minutes, and the color development time of the SmcL and I-inlH-coated plates is 10 minutes.

[0087] As certain embodiments of the present invention, the detection method further includes a judgment step: when the results of ActA, SmcL and I-inlH tests are all negative, it indicates that the host does not suffer from listeriosis; when the ActA test result is positive and the SmcL and I-inlH test results are negative, it indicates that the host is infected with Listeria monocytogenes serotypes other than the 4h serotype; when the ActA and SmcL methods are positive and the I-inlH method is negative, it indicates that the host is infected with the 4h serotype Listeria monocytogenes; when the ActA method is negative and the SmcL and I-inlH methods are positive, it indicates that the host is infected with Listeria ivanovii; when the ActA method is negative and the SmcL and I-inlH methods are positive, it indicates that the host is infected with Listeria monocytogenes; when the ActA, SmcL and I-inlH methods are all positive, it indicates that the host is infected with both Listeria monocytogenes and Listeria ivanovii.

[0088] Other test results except those mentioned above are invalid and require reference to third-party diagnostic methods.

[0089] The present invention also provides the use of ActA, SmcL and I-inlH proteins in the preparation of immunoassay reagents, wherein the use is the use of the ActA, SmcL and I-inlH proteins in the preparation of a detection reagent for detecting Listeria monocytogenes and / or Listeria ivanovii, wherein the immunoassay reagent also includes a standard positive control and a standard negative control, as well as a detection reagent for detecting antigen-antibody reactions between the ActA, SmcL and I-inlH proteins and their antibodies.

[0090] As certain embodiments of the present invention, the detection reagent for detecting Listeria monocytogenes and / or Listeria ivanovii is a detection reagent for distinguishing and detecting Listeria monocytogenes from Listeria ivanovii.

[0091] As certain embodiments of the present invention, the detection reagent for detecting Listeria monocytogenes and / or Listeria ivanovii is a detection reagent for distinguishing and detecting serotype 4h Listeria monocytogenes or other Listeria monocytogenes.

[0092] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0093] It should be understood that the terms used in the examples of the present invention are intended to describe specific embodiments and are not intended to limit the scope of protection of the present invention. In the present specification and claims, unless otherwise expressly indicated, the singular forms "a," "an," and "the" include the plural forms. When numerical ranges are given in the examples, it should be understood that unless otherwise indicated, both endpoints of each numerical range and any value between the two endpoints may be used.

[0094] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition to the specific methods, devices, and materials used in the examples, any prior art methods, devices, and materials similar or equivalent to those in the examples may be used to implement the present invention, based on the knowledge of the prior art by those skilled in the art and the disclosure of this invention.

[0095] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in this technical field and related fields.

[0096] Example 1: Amplification of ActA, SmcL and I-inlH protein encoding genes and construction and expression of expression vectors

[0097] 1. Primer design and synthesis

[0098] Three pairs of specific primers were designed and synthesized based on the gene sequences encoding the ActA, SmcL, and I-inlH proteins published in GenBank: the upstream and downstream primer sequences for amplifying the actA gene are shown in SEQ ID NOs: 7 and 10; the upstream and downstream primer sequences for amplifying the smcL gene are shown in SEQ ID NOs: 8 and 11; and the upstream and downstream primer sequences for amplifying the i-inlH gene are shown in SEQ ID NOs: 9 and 12. The upstream and downstream primers were designed with dual restriction sites, BamHI and HindIII, respectively. The primers were synthesized, purified, and sequenced by Beijing Qingke Biotechnology Co., Ltd.

[0099] 2. PCR amplification using the above primers

[0100] The PCR amplification results, using the Listeria monocytogenes LM4 genome and the Listeria ivanovii XYSL genome as templates, are shown in Figure 1. The PCR amplification reaction system consisted of 50 μL of the amplification system, 25 μL of 2× Primestar max DNA Polymerase, 2 μL of upstream and downstream primers, and 2 μL of template, supplemented with sterile ddH2O to a final volume of 50 μL. The PCR amplification reaction conditions were: 98°C denaturation for 5 minutes followed by cycling; 34 cycles of denaturation at 98°C for 10 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 1 minute; and a final extension at 72°C for 10 minutes.

[0101] 3. Construction of recombinant plasmid

[0102] After the reaction, 50 μL of PCR product was taken for nucleic acid electrophoresis identification, the product was separated by electrophoresis, purified by gel cutting, connected to the linearized pET-30a vector, and transformed into Escherichia coli DH5α competent cells; kanamycin resistance (Kan + ) plates overnight, single white colonies were picked for purification and identification, and plasmids were extracted for double enzyme digestion and electrophoresis detection. The results are shown in Figure 2. The plasmids identified as correct by electrophoresis were sent to Beijing Qingke Bioengineering Co., Ltd. for sequencing.

[0103] The actA protein sequence is shown in SEQ ID NO: 1:

[0104] The smcL protein sequence is shown in SEQ ID NO: 2:

[0105] The i-in1H protein sequence is shown in SEQ ID NO: 3:

[0106] The actA gene sequence is shown in SEQ ID NO: 4:

[0107] The smcL gene sequence is shown in SEQ ID NO: 5:

[0108] The i-in1H gene sequence is shown in SEQ ID NO: 5:

[0109] 4. Construction and expression of expression vector

[0110] The positive recombinant plasmid was transformed into BL21 (DE3) competent cells, and white colonies were picked for purification and identification. The positive single colonies BL21 (DE3) (pET-30a-actA) were inoculated into the culture medium containing Kan + ActA protein was purified using an affinity chromatography column (His-Bind purification kit, purchased from Novagen) using a His-Bind purification kit. rHis-SmcL and rHis-I-inlH were derived from rHis-SmcL and rHis-I-inlH, with the exception of an induction temperature of 15°C and a time of 20 h. SDS-PAGE electrophoresis was then performed, yielding target fusion protein fragments with molecular weights of 97 kDa, 36 kDa, and 35 kDa, respectively, as shown in Figure 3.

[0111] Example 2: Western blot and bioreactivity identification of ActA, SmcL and I-inlH recombinant proteins

[0112] 1. Western blot identification of ActA, SmcL, and I-inlH proteins

[0113] The purified recombinant fusion proteins were subjected to SDS-PAGE electrophoresis and then Western blotted using a His monoclonal antibody solution as the primary antibody and an HRP-labeled goat anti-mouse polyclonal antibody as the secondary antibody. The slides were then developed using ECL ultrasensitive luminescence developer for 2 minutes in the dark, followed by reading. The results showed that the three Listeria recombinant proteins of the present invention reacted with the specific monoclonal antibodies, indicating that the proteins were correctly identified (Figure 4). The purified and identified recombinant proteins were dialyzed and their concentrations were determined.

[0114] 2. Bioreactivity Identification of ActA, SmcL, and I-inlH Proteins

[0115] To verify the distribution of ActA, SmcL, and I-inlH proteins in 4h serotype Listeria monocytogenes, non-4h serotype Listeria monocytogenes, and Listeria ivanovii, the three purified and characterized proteins were loaded onto SDS-PAGE gel wells. After transfer to the membrane and blocking, Western blot reactions were performed using positive polyclonal antibodies against sheep infected with Listeria monocytogenes and / or Listeria ivanovii as primary antibodies, and rabbit anti-sheep enzyme-linked antibodies were used as secondary antibodies for color development. Four different reaction conditions were set: ① serum from sheep infected with a combination of Listeria monocytogenes and Listeria ivanovii; ② serum from sheep infected with 4h Listeria monocytogenes; ③ serum from sheep infected with 1 / 2a serotype 4 Listeria monocytogenes; and ④ serum from sheep infected with Listeria ivanovii. As shown in Figure 5 , the specific distribution of ActA, SmcL and I-inlH proteins in 4h-type Listeria monocytogenes, non-4h-type Listeria monocytogenes and Listeria ivanovii is shown in Table 1 .

[0116] Table 1 Distribution of ActA, SmcL and I-inlH antigens in 4h serotype Listeria monocytogenes, non-4h serotype Listeria monocytogenes and Listeria ivanovii

[0117] Note: “+” indicates existence, and “-” indicates non-existence.

[0118] Example 3: Establishment of an indirect ELISA method for distinguishing between Listeria monocytogenes and Listeria ivanovii infected sheep

[0119] 1. Determination of the optimal antigen coating concentration and enzyme-labeled secondary antibody working concentration

[0120] The optimal coating concentration for the antigen and the optimal dilution of the enzyme-labeled secondary antibody were determined using an ELISA checkerboard assay. ActA, SmcL, and I-inlH proteins were serially diluted in carbonate buffer to various concentrations (10 μg / mL, 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.625 μg / mL, 0.31 μg / mL, and 0.16 μg / mL). 100 μL / well was added and incubated at 4°C for 16 hours, followed by washing with PBST. The next day, 200 μL of PBS containing 2% BSA was added to each well, incubated at 37°C for 2 hours, and washed with PBST. The same aliquots of positive and negative goat sera used to determine the optimal reaction conditions were diluted 1:200 in PBST containing 1% BSA. 100.5 μL of these diluted serum samples were added to the enzyme-labeled wells, incubated in a 37°C waterbath for 2 hours, and washed with PBST. Then, rabbit anti-goat enzyme-labeled secondary antibody was diluted to 1:5000, 1:10000, 1:20000 and 1:40000 respectively. 100 μL of the diluted secondary antibody was added to each well of the ELISA plate, incubated in a 37°C water bath for 1 hour, and washed with PBST. TMB was added for 10 minutes, and the color was stopped with 2M H2SO4 solution and the OD was read on the ELISA reader. 450 Select the positive OD 450 The value is around 1.0 and the OD is negative 450 The value is below 0.2 and the OD is positive. 450 / Negative OD 450 The condition with the larger (P / N) value was taken as the optimal working concentration of coating antigen and rabbit anti-goat enzyme-labeled secondary antibody.

[0121] The results are shown in Figure 6. Considering the cost and dosage of the coating antigen and enzyme-labeled secondary antibody, when the ActA antigen coating concentration was 0.625 μg / mL and the enzyme-labeled secondary antibody dilution ratio was 1:40000, the positive OD 450 The value is closest to 1.0 (1.054); when the SmcL antigen coating concentration is 0.625μg / mL and the enzyme-labeled secondary antibody dilution is 1:10000, the positive OD 450 The value is closest to 1.0 (1.037); when the concentration of I-inlH antigen coating is 1.25μg / mL and the dilution ratio of enzyme-labeled secondary antibody is 1:20000, the positive OD 450The value was closest to 1.0 (1.081). The indirect ELISA reaction was most stable at this time, and the P / N value was large under this condition. Therefore, the optimal antigen coating concentration and optimal enzyme-labeled secondary antibody dilution ratio for indirect ELISA were determined to be 0.625 μg / mL, 1:40,000 (ActA), 0.625 μg / mL, 1:10,000 (SmcL), and 1.25 μg / mL, 1:20,000 (I-inlH), respectively. The secondary antibody concentrations at this time were 50 ng / mL, 200 ng / mL, and 100 ng / mL, respectively.

[0122] 2. Determination of coating conditions

[0123] Based on the optimal reaction conditions determined above, 4 groups of coating conditions were set up and the OD values ​​of each group were compared. 450 The optimal coating conditions were selected based on the time and P / N values. Figure 7 shows that the optimal coating conditions for the ActA indirect ELISA were 37°C for 2 hours, and 4°C for 16 hours for the SmcL and I-inlH indirect ELISAs.

[0124] 3. Determination of blocking solution

[0125] Based on the optimal reaction conditions determined above, four sets of blocking buffer conditions were set, and the blocking buffer with the highest P / N value was selected as the optimal blocking buffer. The results are shown in Figure 8. For the ActA indirect ELISA, PBS containing 5% BSA was the optimal blocking buffer; for the SmcL and I-inlH indirect ELISAs, PBS containing 5% skim milk was the optimal blocking buffer.

[0126] 4. Determination of closure time

[0127] Based on the optimal reaction conditions determined above, four different blocking times were set, and the condition with the highest average P / N ratio was selected as the optimal blocking time. The results, as shown in Figure 9, showed that the optimal blocking time for the ActA indirect ELISA method was 2.5 hours, while the optimal blocking time for the SmcL and I-inlH indirect ELISA methods was 1.5 hours.

[0128] 5. Determination of serum primary antibody incubation time

[0129] Based on the optimal reaction conditions determined above, positive and negative control sera were diluted at a serum dilution factor of 1:200 and incubated at 37°C for 1, 1.5, 2, and 2.5 hours, respectively. The results are shown in Figure 10. For the ActA indirect ELISA, an incubation time of 1 hour was selected as the optimal primary antibody incubation time; for the SmcL and I-inlH indirect ELISAs, an incubation time of 2.5 hours was selected as the optimal primary antibody incubation time.

[0130] 6. Determination of enzyme-labeled antibody reaction time

[0131] Based on the optimal reaction conditions determined above, different HRP-rabbit anti-goat IgG enzyme-labeled antibody incubation times were set at 30, 45, 60, and 75 minutes. The results, as shown in Figure 11, show that 30 minutes was the optimal secondary antibody reaction time for the ActA indirect ELISA method, while 60 minutes was the optimal secondary antibody reaction time for the SmcL and I-inlH indirect ELISA methods.

[0132] 7. Determination of substrate color development time

[0133] Based on the optimal reaction conditions determined above, four different TMB development times were set. The results, as shown in Figure 12, showed that 5 minutes was the optimal development time for the ActA indirect ELISA method, while 10 minutes was the optimal development time for the SmcL and I-inlH indirect ELISA methods.

[0134] Example 4: Assembly of Sheep Listeriosis ELISA Kit and Determination of Threshold

[0135] The ELISA kit consists of the following: 3 antigen-coated plates (1 each of ActA, SmcL, and I-inlH proteins), 800 μL each of 1:200 diluted positive control serum and negative control serum, 50 mL of antibody diluent (1x), 1 mL of 1:1000 diluted rabbit anti-sheep enzyme-labeled antibody (diluted with antibody diluent to the optimal ratio for each method when used), 60 mL of PBST wash solution (20×), 30 mL of TMB substrate developer, 20 mL of 2 mol / L H2SO4 stop solution, and 1 instruction manual.

[0136] The threshold values ​​of the ELISA method using ActA, SmcL, and I-inlH as coating antigens were determined according to the ELISA reaction conditions determined in Example 3. Specifically, 94 negative sera tested against Listeria monocytogenes using a third-party kit were tested, and the positive and negative control test values ​​were recorded as P and N. The average of the 94 negative serum test results was calculated. and standard deviation SD, to obtain the critical value Cut-off value = (LN) / (PN), the sample is tested for OD 450 The value is recorded as S. If S / P = (SN) / (PN) > Cut-off value, it is judged as positive serum, otherwise it is negative serum.

[0137] The results are shown in Table 2(a), (b) and (c). The average values ​​of the ActA-iELISA method were The cut-off value was calculated to be 0.104. The average value of the SmcL-iELISA method was 0.157, the standard deviation SD was 0.024, and the cut-off value was calculated to be 0.104. The cut-off value was calculated to be 0.110, with a standard deviation of 0.018. The average value of the I-inlH-iELISA method was The cut-off value is 0.137.

[0138] Table 2 (a) ActA indirect ELISA threshold determination results

[0139] Table 2(b) SmcL indirect ELISA threshold determination results

[0140] Table 2(c) Threshold determination results of I-inlH indirect ELISA

[0141] Example 5: Repeatability test of sheep listeriosis ELISA kit

[0142] Repeatability test was performed according to the indirect ELISA method established in Example 3. Intra-batch repeat stability test was performed by using the same batch of purified protein coated ELISA plate to test the same 6 sheep sera for 3 consecutive days; inter-batch repeat stability test was performed by using 3 batches of purified protein coated ELISA plate to test the same 6 sheep sera at the same time. and standard deviation SD, and finally use The coefficient of variation (CV%) of each sample was calculated.

[0143] As shown in Tables 3(a), (b), and (c), the intra-assay CV for the ActA-iELISA assay ranged from 2.45% to 6.74%, and the inter-assay CV ranged from 3.15% to 8.28%. The intra-assay CV for the SmcL-iELISA assay ranged from 1.42% to 5.54%, and the inter-assay CV ranged from 5.25% to 9.61%. The intra-assay CV for the I-inlH-iELISA assay ranged from 1.96% to 7.23%, and the inter-assay CV ranged from 4.46% to 12.48%. These results demonstrate that the ELISA assay has low variability and good repeatability.

[0144] Table 3 (a) ActA-iELISA repeatability test results

[0145] Table 3(b) SmcL-iELISA repeatability test results

[0146] Table 3 (c) I-inlH-iELISA repeatability test results

[0147] Example 6: Practicality test of sheep listeriosis ELISA kit

[0148] 1. Dynamic changes in ActA and SmcL antibody levels

[0149] 4h serotype Listeria monocytogenes strain XYSN was added at 1.5×10 10 Five Hu sheep were orally infected with a dose of CFU. The indirect ELISA method established in Example 3 was used to detect ActA and SmcL antibodies in the serum, and the pattern of antibody production of ActA and SmcL in the sheep serum was analyzed. The time of ActA and SmcL antibody production in Hu sheep after infection with XYSN is shown in Figures 13A and 13B. All Hu sheep tested positive by the ActA-iELISA method 6 days after infection, that is, they had listeriosis. The antibody levels mostly reached a peak around the 18th day, and anti-ActA antibodies were still at a high level 32 days after infection. The results show that the ActA and SmcL detection components in the kit can diagnose listeriosis in the early stage of host infection and for a long time after infection.

[0150] 2. Dynamic changes in I-inlH antibody levels

[0151] Listeria monocytogenes strain XYSL was isolated at 3×10 9 Six-week-old BALB / c mice were orally infected with a dose of 100 CFU. I-in1H antibodies in serum were detected using an indirect ELISA method established according to Example 3. As shown in Figure 13C, BALB / c mice tested positive for listeriosis six days after infection, with antibody levels reaching their highest on day 16. Antibodies against I-in1H remained at high levels 30 days after infection. These results demonstrate that the I-in1H detection component in the kit can diagnose listeriosis both early in the host infection and long after infection.

[0152] Example 7: Thermal stability of the ELISA test kit for sheep listeriosis

[0153] The antigen-coated plates were heat-damaged by coating and sealing according to the optimal conditions determined in Example 3, then oven-dried, vacuum-packed in aluminum foil, and stored in a 37°C incubator for 6, 7, and 8 days. The rabbit anti-sheep enzyme-labeled antibody was heat-damaged by diluting it 1:50 with Candor's HRP-Protector™ protectant, sealing it, and storing it in a 37°C incubator for 6, 7, and 8 days. The serum controls were heat-damaged by storing the diluted positive and negative control sera in a 37°C incubator for 6, 7, and 8 days. The intra-assay coefficients of variation of the three control sera were determined using an indirect ELISA method established in Example 3. Positive reference refers to the positive reference, and negative reference refers to the negative reference. The test results, shown in Tables 4(a), (b), and (c), indicate that the intra-assay coefficients of variation of all components in the kit under heat-damage conditions were less than 15%, indicating that the entire kit meets the thermal stability requirements. Its shelf life at 4°C is predicted to be greater than 8 months.

[0154] Table 4 (a) Thermal stability analysis of ActA kit

[0155] Table 4(b) Thermal stability analysis of SmcL kit

[0156] Table 4 (c) Thermal stability analysis of I-inlH kit

[0157] Example 8: Specificity and sensitivity of the sheep listeriosis ELISA kit

[0158] The specificity and sensitivity of the three detection components in the indirect ELISA test kit established according to Example 3 are calculated and shown in Tables 5(a), (b), and (c). The ActA test kit was used to test 65 sera from sheep infected with Listeria monocytogenes and 92 uninfected sera. 61 positive samples were detected, resulting in a sensitivity of 93.85%; 87 negative samples were detected, resulting in a specificity of 94.57%. The SmcL test kit was used to test 70 sera from sheep infected with 4h-type Listeria monocytogenes or Listeria ivanovii and 92 uninfected sera. 66 positive samples were detected, resulting in a sensitivity of 94.29%; 88 negative samples were detected, resulting in a specificity of 95.65%. The I-inlH test kit was used to test 15 sera from sheep infected with Listeria ivanovii and 92 uninfected sera. 15 positive samples were detected, resulting in a sensitivity of 100%; 87 negative samples were detected, resulting in a specificity of 94.57%. All sera were sourced and tested using a third-party assay. The results demonstrated that the components of the ELISA kit had good sensitivity and specificity.

[0159] Table 5 (a) Sensitivity and specificity analysis of ActA kit

[0160] Table 5(b) Sensitivity and specificity analysis of the SmcL kit

[0161] Table 5(c) Sensitivity and specificity analysis of I-inlH kit

[0162] Example 9: Compliance test of sheep listeriosis ELISA kit

[0163] The sheep listeriosis ELISA kit described in the present invention was used to test several clinical sheep sera with a clear background stored in the laboratory. The test results were compared with those of the imported Listeria Sheep Test Kit (Diatheva) based on the LLO antigen. The results are shown in Tables 6(a), (b), and (c). For the ActA kit, a total of 79 clinical sheep serum samples were tested, and the test results of 75 samples were consistent, with an overall compliance rate of 94.94%; for the SmcL kit, a total of 79 clinical sheep serum samples were tested, and the test results of 72 samples were consistent, with an overall compliance rate of 91.14%; for the I-inlH kit, a total of 50 clinical sheep serum samples were tested, and the test results of 46 samples were consistent, with an overall compliance rate of 92%.

[0164] Table 6 (a) Comparison of ActA kit and Diatheva kit

[0165] Table 6 (b) Comparison of SmcL kit and Diatheva kit

[0166] Table 6 (c) Comparison of I-inlH kit and Diatheva kit

[0167] Example 10: Application of ELISA Detection Kit for Sheep Listeriosis

[0168] Using the indirect ELISA test kit described herein, a traceability investigation was conducted on the sheep farm described in Example 10. A total of 40 sheep serum samples were collected from a domestic sheep farm for testing. The results showed that three sheep tested positive for ActA and SmcL antibodies, indicating infection with the 4h serotype of Listeria monocytogenes; one sheep tested positive for ActA antibodies, indicating infection with a non-4h serotype of Listeria monocytogenes; 12 sheep tested positive for SmcL and I-inlH antibodies, indicating infection with Listeria ivanovii; and 15 sheep tested positive for ActA, SmcL, and I-inlH antibodies, indicating mixed infection with Listeria monocytogenes and Listeria ivanovii. The overall antibody positivity rate reached 77.5%. In addition, four strains of 4h serotype monocytogenes Listeria and one strain of Listeria ivanovii were isolated from the wipe samples and environmental samples collected from the sheep farm, indicating that the positive rate of pathogenic Listeria infection in the sheep farm is high, and prevention, control and disinfection and sterilization work should be done well.

[0169] Table 7 ELISA kit for sheep listeriosis detection of sheep serum samples from a certain place in East China

[0170] The inventors previously used bioinformatics software to predict the hydrophilicity, surface polarity, antigenicity, and secondary structural characteristics of the I-in1H protein, and conducted predictive analysis of the potential antigenic epitope regions of the protein. The inventors found that truncated fragments of the protein possessed good conservation and immunogenicity. After constructing and successfully expressing ActA, SmcL, and I-in1H proteins, the specific distribution of these three antigens in 4h serotype Listeria monocytogenes, other serotypes of Listeria monocytogenes, and Listeria ivanovii was verified. Based on this premise, the inventors studied and designed an indirect ELISA kit for distinguishing between infections with Listeria monocytogenes and Listeria ivanovii species of the genus Listeria monocytogenes. The kit of the present invention can effectively distinguish between sheep infected with 4h serotype Listeria monocytogenes, non-4h serotypes of Listeria monocytogenes, Listeria ivanovii, or mixed infections of Listeria monocytogenes and Listeria ivanovii, and has good specificity, sensitivity, and repeatability, facilitating large-scale detection of listeriosis in sheep farms.

[0171] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form or substance. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention. Any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the spirit and scope of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. An indirect ELISA kit for detecting Listeria monocytogenes and / or Listeria ivanovii, characterized in that: The kit comprises carriers coated with ActA, SmcL and I-inlH proteins respectively, a standard positive control and a standard negative control, and a detection reagent for detecting antigen-antibody reaction between ActA, SmcL and I-inlH proteins and their antibodies.

2. The indirect ELISA kit according to claim 1, characterized in that The amino acid sequence of the ActA protein is shown in SEQ ID NO.1, the amino acid sequence of the SmcL protein is shown in SEQ ID NO.2, and the amino acid sequence of the I-inlH protein is shown in SEQ ID NO.

3.

3. The indirect ELISA kit according to any one of claims 1 to 2, characterized in that: The antibodies are respectively monoclonal antibodies or polyclonal antibodies against ActA protein, monoclonal antibodies or polyclonal antibodies against SmcL protein, and monoclonal antibodies or polyclonal antibodies against I-inlH protein; The detection reagent is a secondary antibody labeled with a marker and a substrate color developing solution; Preferably, the label is horseradish peroxidase (HRP), alkaline phosphatase, or biotin; Preferably, the substrate color developing solution is 3,3',5,5'-tetramethylbenzidine (TMB).

4. The indirect ELISA kit according to any one of claims 1 to 3, characterized in that: The indirect ELISA kit sample diluent, washing solution, color developing solution and stop solution; The sample diluent is a PBST solution containing 1% BSA, the washing solution is a PBST solution with a pH of 7.2 and containing 0.05% Tween 20, and the stop solution is a 2 mol / L H2SO4 solution.

5. The indirect ELISA kit according to any one of claims 1 to 4, characterized in that: The standard positive controls include positive sera from hosts infected with serotype 4h monocytogenes, other monocytogenes, or Listeria ivanovii, respectively; The standard negative control includes negative serum from a host not infected with serotype 4h monocytogenes Listeria monocytogenes, other Listeria monocytogenes, or Listeria ivanovii; Or the carriers coated with ActA, SmcL and I-inlH proteins respectively include ELISA plates.

6. A method for preparing the indirect ELISA kit according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: (1) Coating: ActA, SmcL and I-inlH proteins are coated on an ELISA plate respectively, wherein the amino acid sequence of the ActA protein is shown in SEQ ID NO.1, the amino acid sequence of the SmcL protein is shown in SEQ ID NO.2, and the amino acid sequence of the I-inlH protein is shown in SEQ ID NO.3; (2) Blocking: using a blocking solution to block the ELISA plate coated with ActA, SmcL and I-inlH proteins in step (1); (3) preparing a standard positive control and a standard negative control; preferably, the standard positive control includes: the standard negative control; (4) preparing a secondary antibody labeled with a marker and a substrate color development solution; The indirect ELISA kit is assembled with the ELISA plate prepared in step (2), the standard positive control prepared in step (3), the standard negative control, the labeled secondary antibody prepared in step (4), and the substrate color development solution.

7. The method according to claim 6, characterized in that In step (1), the ActA, SmcL and I-inlH proteins were dissolved in CBS coating solution and coated on ELISA plates at final concentrations of 0.625, 0.625 and 1.25 μg / ml, respectively. The ActA coated plates were coated at 37° C. for 2 hours, and the SmcL and I-inlH coated plates were coated at 4° C. for 16 hours. Or in step (2), the ActA coated plate was blocked with PBS containing 5% BSA for 2.5 h, and the SmcL and I-inlH coated plates were blocked with PBS containing 5% skim milk for 1.5 h.

8. The method according to any one of claims 5 to 6, characterized in that: The method further comprises preparing a sample diluent, a washing solution, a color developing solution and a stop solution; Preferably, the label is horseradish peroxidase (HRP), alkaline phosphatase, or biotin; Preferably, the sample diluent is a PBST solution containing 1% w / v BSA; Preferably, the washing solution is a PBST solution with a pH of 7.2 and containing 0.05% w / v Tween 20. Preferably, the substrate color developing solution is 3,3',5,5'-tetramethylbenzidine (TMB); Preferably, the stop solution is a 2 mol / L H2SO4 solution.

9. The method according to any one of claims 5 to 7, characterized in that: The optimal coating concentration of ActA protein was 0.625 μg / mL, the coating condition was 37°C for 2 h, the blocking condition was PBS solution containing 5% BSA for 2.5 h, and the working concentration of the enzyme-labeled secondary antibody was 1:40000; The coating concentration of the SmcL protein was 0.625 μg / mL, the coating condition was 16 h at 4°C, the blocking condition was 1.5 h with a PBS solution containing 5% skimmed milk powder, and the working concentration of the enzyme-labeled secondary antibody was 1:10000; The coating concentration of the I-inlH protein was 1.25 μg / mL, the coating conditions were 16 h at 4° C., the blocking conditions were 1.5 h of blocking with a PBS solution containing 5% skimmed milk powder, and the working concentration of the enzyme-labeled secondary antibody was 1:20,000.

10. Use of the indirect ELISA kit according to any one of claims 1 to 5, wherein the use is use of the indirect ELISA kit in preparing a detection reagent for detecting Listeria monocytogenes and / or Listeria ivanovii; Preferably, the detection reagent for detecting Listeria monocytogenes and / or Listeria ivanovii is a detection reagent that can distinguish and detect Listeria monocytogenes and Listeria ivanovii with high specificity; Preferably, the detection reagent for detecting Listeria monocytogenes and / or Listeria ivanovii is a detection reagent that can distinguish and detect serotype 4h Listeria monocytogenes or other Listeria monocytogenes with high specificity.

11. The method for detecting Listeria monocytogenes and / or Listeria ivanovii according to the indirect ELISA kit according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: (1) Incubation of test samples: dilute the test samples with diluent, incubate the diluted test samples on ActA coated plates, SmcL coated plates, and I-inlH coated plates, respectively, and add the respective standard negative controls and standard positive controls; (2) Secondary antibody incubation: adding secondary antibody to the ActA coated plate, SmcL coated plate, and I-inlH coated plate to which serum was added in step (1), and incubating; (3) Color development: adding the detection reagent to the coated plate incubated with the secondary antibody in step (2), color development, then adding the stop solution to stop, and reading the absorbance value at OD450nm; (4) Result determination: The ELISA method uses the S / P ratio for determination, and the formula is S / P = (sample OD450nm value - negative control OD450nm average value) / (positive control OD450nm average value - negative control OD450nm average value); for ActA detection results, S / P ratio > critical value 0.104 is judged as positive, otherwise it is judged as negative; for SmcL detection results, S / P ratio > critical value 0.110 is judged as positive, otherwise it is judged as negative; for I-inlH detection results, S / P ratio > critical value 0.137 is judged as positive, otherwise it is judged as negative.

12. The detection method according to claim 11, characterized in that The test samples in step (1) were incubated on ActA coated plates at 37°C for 1 hour, and on SmcL and I-inlH coated plates at 37°C for 2.5 hours; Or the test sample in step (1) is serum; Or in the step (2), 100 μl of a secondary antibody diluted 1:20,000 is added to the ActA coated plate and the SmcL coated plate of the step (1) and incubated for 60 min; 100 μl of a secondary antibody diluted 1:10,000 is added to the I-inlH coated plate and incubated for 60 min; Or in the step (3), the color development time of the ActA coated plate is 5 minutes, and the color development time of the SmcL and I-inlH coated plates is 10 minutes.

13. The detection method according to any one of claims 11 to 12, characterized in that: The detection method also includes a determination step: When the results of ActA, SmcL, and I-inlH tests are all negative, it means that the host does not have listeriosis; When the ActA test result is positive and the SmcL and I-inlH test results are negative, it means that the host is infected with Listeria monocytogenes serotypes other than serotype 4h; When the results of the ActA and SmcL methods were positive and the results of the I-inlH method were negative, it indicated that the host was infected with 4h serotype Listeria monocytogenes; When the ActA method test is negative and the SmcL and I-inlH methods test are positive, it means that the host is infected with Listeria ivanovii; When ActA, SmcL and I-inlH methods all test positive, it means that the host is infected with Listeria monocytogenes and Listeria ivanovii at the same time.

14. Use of ActA, SmcL and I-inlH proteins in the preparation of immunoassay reagents, characterized in that: The application is the use of the ActA, SmcL and I-inlH proteins in the preparation of a detection reagent for detecting Listeria monocytogenes and / or Listeria ivanovii, and the immunodetection reagent also includes a standard positive control and a standard negative control, and a detection reagent for detecting antigen-antibody reactions between ActA, SmcL and I-inlH proteins and their antibodies; Preferably, the detection reagent for detecting Listeria monocytogenes and / or Listeria ivanovii is a detection reagent that can distinguish and detect Listeria monocytogenes and Listeria ivanovii with high specificity; Preferably, the detection reagent for detecting Listeria monocytogenes and / or Listeria ivanovii is a detection reagent that can distinguish and detect serotype 4h Listeria monocytogenes or other Listeria monocytogenes with high specificity.

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