ELISA kit for distinguishing listeria monocytogenes and / or listeria ivanovii infection as well as preparation method and application of ELISA kit for distinguishing listeria monocytogenes and / or listeria ivanovii infection

Abstract

The invention discloses an indirect ELISA (Enzyme-Linked Immunosorbent Assay) detection kit for distinguishing host infected Listeria monocytogenes and / or Listeria ivanovii. The coating antigens are respectively a recombinant listeria monocytogenes surface protein ActA, a serotype 4h listeria monocytogenes and listeria ivanovii common outer membrane protein SmcL and a listeria ivanovii specific outer membrane protein I-inlH, indirect ELISA methods are respectively established based on the three antigens, the three detection methods are combined to detect the content of the listeria monocytogenes, and the content of the listeria ivanovii can be detected. The levels of the ActA antibody, the SmcL antibody and the I-inlH antibody in a clinical serum sample can be effectively monitored, and the detection on the listeria monocytogenes caused by the listeria monocytogenes and / or the listeria ivanovii is realized. The kit is relatively high in sensitivity, strong in specificity and good in stability, and an effective method is provided for epidemiological investigation and diagnosis of listeria monocytogenes.

Description

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 (hereinafter referred to as Lm) and Listeria ivanovii (hereinafter referred to as Liv) are the main pathogenic species of Listeria, which can cause listeriosis in humans and animals. Among them, Listeria monocytogenes infects animals and causes serious economic losses to farms. The Listeria monocytogenes carried by animals enters the food chain through the slaughtering and processing links, and reaches the table along the animal product processing, transportation, and sales links, causing human infection, with a mortality rate of up to 20%-30%. Most listeriosis is caused by serotypes 1 / 2b and 4b of lineage I and serotype 1 / 2a of lineage II. The virulent strain of ST626 isolated from the outbreak of listeriosis in sheep farms belongs to the newly named 4h serotype monocytogenes. Listeria ivanovii mainly infects ruminants. Sheep flocks are susceptible hosts of Listeria in nature, and sheep listeriosis has brought huge economic losses to the breeding industry. In recent years, there have been reports of cases of human gastroenteritis and sepsis caused by Listeria monocytogenes, which require high attention.

[0003] Strengthening the monitoring of listeriosis in the breeding process can detect infected animals at the farm stage and take timely measures to prevent sick livestock from entering the food industry chain, which is of great significance to public health and safety. The "gold standard" for the diagnosis of listeriosis is the isolation of pathogens. However, this method is time-consuming, and the test results are often inaccurate due to the short existence time of live bacteria and irregular excretion of bacteria. Serological detection methods are often used for auxiliary diagnosis of infectious diseases, which is a supplement and support for traditional bacterial isolation and culture methods. Among them, the ELISA method has the advantages of simple operation, fast speed, and high-throughput screening of large quantities of samples. It is widely used in epidemiological surveys of sheep farms and clinical immune monitoring. At present, there is a commercial diagnostic kit for the detection of listeriosis, which is an indirect ELISA method based on listerolysin O (Listerolysin, LLO). This kit cannot distinguish between infections caused by Listeria monocytogenes and Listeria ivanovii, and there is a cross-reaction with serum antibodies after infection with other genera such as Streptococcus.

[0004] ActA is an important virulence factor of Listeria monocytogenes, located on the cell wall surface and has been shown to have strong immunogenicity. SmcL protein is an outer membrane protein common to 4h-type Listeria monocytogenes and Listeria ivanovii. I-inlH protein is an outer membrane protein unique to 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. Summary of the invention

[0006] In order to solve the deficiencies in the prior art, the present invention selects the surface protein ActA of Listeria monocytogenes, the outer membrane protein SmcL shared by 4h type 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, which can not only detect whether a sample is infected with Listeria monocytogenes, but also distinguish whether the 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.

[0007] 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 reaction between ActA, SmcL and I-inlH proteins and antibodies thereof.

[0008] The present invention is the first to detect host infection with Listeria monocytogenes and / or Listeria ivanovii with high specificity and sensitivity by combining ActA, SmcL and I-inlH antigens. The inventors have discovered for the first time that these three proteins have good conservation and antigenicity, and can be used as coating antigens to prepare indirect ELISA kits. In view of this, the inventors have studied and designed an indirect ELISA kit for distinguishing between Listeria monocytogenes and Listeria ivanovii species infections.

[0009] 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 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; (4) preparing an enzyme-labeled secondary antibody and a color developing solution; assembling the ELISA plate prepared in step (2), the standard positive control and the standard negative control prepared in step (3), the enzyme-labeled secondary antibody and the color developing solution prepared in step (4) into the indirect ELISA kit.

[0010] The 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 of the Listeria genus with high specificity and sensitivity.

[0011] The present invention further 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. The indirect ELISA kit of the present invention can simultaneously and distinguish between detecting Listeria monocytogenes and / or Listeria ivanovii, and can also distinguish between infection caused by serotype 4h Listeria monocytogenes and infection caused by other Listeria monocytogenes.

[0012] As described above, the indirect ELISA kit, preparation method and application for detecting Listeria monocytogenes and / or Listeria ivanovii have the following beneficial effects:

[0013] 1) The present invention cloned and expressed the recombinant protein I-inlH of Listeria monocytogenes for the first time. Immunoblotting showed that the recombinant ActA, SmcL and I-inlH proteins all had good reactogenicity, and after the 4h-type monocytogenes Listeria monocytogenes XYSN infected Hu sheep, it could induce the production of specific antibodies against ActA and SmcL proteins; after the Listeria monocytogenes strain XYSL infected mice, it could produce specific antibodies against I-inlH, indicating that the ActA, SmcL and I-inlH proteins all had good immunogenicity.

[0014] 2) The detection method and kit for detecting antibodies to Listeria monocytogenes and / or Listeria ivanovii of the present invention can diagnose whether the host suffers from listeriosis and can also determine the species of Listeria that the host is infected with. The kit can distinguish between Listeria monocytogenes and / or Listeria ivanovii infections in the host with high sensitivity, strong specificity and good stability. The present invention can minimize the occurrence rate of false positives and avoid interference with the detection caused by other bacterial antigens of the Listeria genus. Compared with the indirect ELISA detection method based on LLO antigen, which is currently widely used in veterinary clinics, the present invention has higher specificity.

[0015] 3) The establishment of this method fills the gap in serological detection methods for distinguishing between Listeria monocytogenes and Listeria ivanovii infections, and can be used for the investigation of Listeria epidemic patterns in host fields and clinical immune monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, showing exemplary embodiments of the invention and together with the description serve to explain the principles of the invention.

[0017] Figure 1 A, B and C are schematic diagrams of PCR amplification of 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;

[0018] Figure 2 A, B and C are schematic diagrams of double enzyme digestion identification of recombinant plasmids inserted with 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;

[0019] Figure 3 A, B and C are schematic diagrams of induced expression of 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;

[0020] Figure 4 A, 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;

[0021] Figure 5 A, B, C and D are schematic diagrams of in vitro bioreactivity identification of 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;

[0022] Figure 6 A, B and C are the effects of different antigen coating concentrations and enzyme-labeled secondary antibody dilutions on the OD450 value of positive serum in the ActA, SmcL and I-inlH indirect ELISA methods, respectively;

[0023] Figure 7 A, B and C are the effects of different coating conditions on P / N values ​​in the ActA, SmcL and I-inlH indirect ELISA methods, respectively;

[0024] Figure 8 A, B and C are the effects of different blocking solutions on P / N values ​​in the ActA, SmcL and I-inlH indirect ELISA methods, respectively;

[0025] Fig. 9 A, B and C are the effects of different blocking times on P / N values ​​in the ActA, SmcL and I-inlH indirect ELISA methods, respectively;

[0026] Fig.10 A, B and C are the effects of different incubation times of serum primary antibodies on P / N values ​​in the ActA, SmcL and I-inlH indirect ELISA methods, respectively;

[0027] Fig.11 A, B and C are the effects of different enzyme-labeled secondary antibody reaction times on P / N values ​​in the ActA, SmcL and I-inlH indirect ELISA methods, respectively;

[0028] Fig.12 A, B and C are the effects of different substrate color development times on P / N values ​​in the ActA, SmcL and I-inlH indirect ELISA methods, respectively;

[0029] Fig.13A, B and C are schematic diagrams of the changes in serum IgG antibodies over time after 4h-type monocytogenes strain XYSN infected Hu sheep and Listeria ivanovii strain XYSL infected mice detected by indirect ELISA methods of ActA, SmcL and I-inlH, respectively. DETAILED DESCRIPTION

[0030] The present invention is described in detail below:

[0031] The present invention provides an indirect ELISA kit for detecting Listeria monocytogenes and / or Listeria ivanovii, the kit comprising carriers coated with ActA, SmcL and I-inlH proteins, standard positive control and standard negative control, and a detection reagent for detecting antigen-antibody reaction between ActA, SmcL and I-inlH proteins and their antibodies. Listeria monocytogenes is a Gram-positive small rod without spores and capsules. It can be divided into 11 serotypes according to the bacterial body and flagella antigens. It was isolated from naturally infected rabbits in 1926 and named as Monocytogenes bacillus because of the increase of mononuclear cells in sick animals. It is the main pathogenic bacteria that cause animal and human diseases.

[0032] 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.

[0033] ActA protein is an important virulence factor of Listeria monocytogenes, located on the cell wall surface and has been shown to have strong immunogenicity.

[0034] SmcL protein is an outer membrane protein common to Listeria monocytogenes and Listeria ivanovii.

[0035] The I-inlH protein is an outer membrane protein unique to Listeria monocytogenes.

[0036] 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 the activity of the enzyme. During the determination of the present invention, the specimen to be tested (the antibody to be measured 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 a washing method. Then the enzyme-labeled antibody is added and also bound to the solid phase carrier through the reaction. At this time, the amount of enzyme on the solid phase is in a certain ratio to the amount of the substance to be tested in the specimen. After the substrate of the enzyme reaction is added, the substrate is catalyzed by the enzyme to become a colored product. The amount of the product is directly related to the amount of the substance to be tested in the specimen, so qualitative or quantitative analysis can be performed based on the depth of the color.

[0037] The present invention has determined for the first time that the three proteins, ActA protein, SmcL protein, and I-inlH protein, are immunogenic and can be used for corresponding pathogen infection detection. Their combined use can simultaneously and distinguish between the detection of Listeria monocytogenes and / or Listeria ivanovii, and can also distinguish between serotype 4h Listeria monocytogenes infection and infection caused by other Listeria monocytogenes.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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, monoclonal antibodies or polyclonal antibodies against I-inlH protein; the detection reagents are secondary antibodies labeled with markers, and substrate colorimetric solution.

[0044] As certain embodiments of the present invention, the label is horseradish peroxidase (HRP), alkaline phosphatase, or biotin.

[0045] As certain embodiments of the present invention, the substrate color developing solution is 3,3',5,5'-tetramethylbenzidine (TMB).

[0046] As certain embodiments of the present invention, the indirect ELISA kit comprises a sample diluent, a washing solution, a color developing solution and a stop solution.

[0047] 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 a pH of 7.2 containing 0.05% Tween 20, and the stop solution is a 2 mol / L H2SO4 solution.

[0048] As certain embodiments of the present invention, the standard positive control includes positive sera from hosts infected with serotype 4h monocytogenes Listeria, other monocytogenes Listeria, or Listeria ivanovii, respectively.

[0049] As certain embodiments of the present invention, the standard negative control includes negative serum from a host that is not infected with serotype 4h Listeria monocytogenes, other Listeria monocytogenes, or Listeria ivanovii.

[0050] As certain embodiments of the present invention, the carriers respectively coated with ActA, SmcL and I-inlH proteins include ELISA plates.

[0051] The present invention also provides a method for preparing the indirect ELISA kit, the method comprising the following steps:

[0052] (1) Coating: coating ActA, SmcL and I-inlH proteins 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 developing solution; assembling 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 developing solution into the indirect ELISA kit.

[0053] As certain embodiments of the present invention, the ActA, SmcL and I-inlH proteins in step (1) are dissolved in CBS coating solution, respectively, and coated on ELISA plates 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.

[0054] As certain embodiments of the present invention, the coated antigen may be pre-coated on an ELISA plate, or a blank ELISA plate and a detection antigen may be provided, and the operator may coat the detection antigen on the ELISA plate using a prescribed method before detection.

[0055] As certain embodiments of the present invention, the antigen coating solution is a 0.05 mol / L carbonate (CBS) buffer at pH 9.6 containing ActA, SmcL or I-inlH protein, respectively.

[0056] 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.

[0057] 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.

[0058] As certain embodiments of the present invention, the label is horseradish peroxidase (HRP), alkaline phosphatase, or biotin.

[0059] As certain embodiments of the present invention, the enzyme-labeled secondary antibody is a rabbit anti-goat IgG antibody labeled with horseradish peroxidase.

[0060] As certain embodiments of the present invention, the sample diluent is a PBST solution containing 1% w / v BSA.

[0061] 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.

[0062] As certain embodiments of the present invention, the color developing solution is 3,3',5,5'-tetramethylbenzidine (TMB).

[0063] As certain embodiments of the present invention, the stop solution is a 2 mol / L H2SO4 solution.

[0064] As certain embodiments of the present invention, the optimal coating concentration of ActA protein is 0.625 μg / mL, the coating condition is 37° C. for 2 h, the blocking condition is PBS solution containing 5% BSA for 2.5 h, and the working concentration of the enzyme-labeled secondary antibody is 1:40,000.

[0065] 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 of blocking with PBS solution containing 5% skimmed milk powder, and the working concentration of the enzyme-labeled secondary antibody is 1:10000.

[0066] 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.

[0067] 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.

[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 serotype 4h Listeria monocytogenes or other Listeria monocytogenes.

[0069] 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:

[0070] (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;

[0071] (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;

[0072] (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;

[0073] (4) Result determination: The ELISA method uses the S / P ratio for determination, and the formula is S / P = (sample OD450 nm value - negative control OD450 nm average value) / (positive control OD450 nm average value - negative control OD450 nm 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. 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:

[0074] (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 strain as a template to clone a truncated smcL and i-inlH gene fragments;

[0075] (b) separating the nucleic acid fragments of the recombinant protein by electrophoresis, recovering and purifying the nucleic acid fragments by gel, and connecting them with the prokaryotic expression vector pET-30a of Escherichia coli to obtain recombinant plasmids pET-30a-actA, pET-30a-smcL and pET-30a-i-inlH; and transforming DH5α competent cells to obtain prokaryotic recombinant strains; extracting the plasmids for double enzyme digestion electrophoresis detection, and sequencing and analyzing the recombinant plasmids identified as correct by electrophoresis;

[0076] (c) transforming the recombinant plasmid into Escherichia coli BL21 (DE3) competent cells for prokaryotic expression to obtain three recombinant proteins, namely, rHis-ActA protein of Listeria monocytogenes and rHis-SmcL and rHis-I-inlH proteins of Listeria ivanovii;

[0077] (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.

[0078] As certain embodiments of the present invention, in step (a), the sequences of the primer pairs used for amplification are shown as SEQ ID NOs. 7, 8, 9, 10, 11 and 12.

[0079] In the ELISA test of specific serum samples, the best coating concentration of ActA antigen is 0.625μg / mL, the best coating condition is 37℃ coating for 2h, the best blocking condition is blocking with PBS solution containing 5% BSA for 2.5h, the best serum action time is 1h, the best enzyme-labeled secondary antibody working concentration is 1:40000, the best enzyme-labeled secondary antibody action time is 30min, and the best substrate color development time is 5min. Under the above conditions, when S / P=(SN) / (PN)>0.104, it is judged as ActA positive, otherwise it is negative.

[0080] In the ELISA test of specific serum samples, the best coating concentration of SmcL antigen is 0.625μg / mL, the best coating condition is 16h at 4℃, the best blocking condition is 1.5h with PBS solution containing 5% skim milk powder, the best serum action time is 2.5h, the best enzyme-labeled secondary antibody working concentration is 1:10000, the best enzyme-labeled secondary antibody action time is 60min, and the best substrate color development time is 10min. Under the above conditions, when S / P=(SN) / (PN)>0.110, it is judged as SmcL positive, otherwise it is negative.

[0081] In the ELISA test of specific serum samples, the optimal coating concentration of I-inlH antigen is 1.25μg / mL, the optimal coating condition is 16h at 4°C, the optimal blocking condition is 1.5h with PBS solution containing 5% skim milk powder, the optimal serum action time is 2.5h, the optimal enzyme-labeled secondary antibody working concentration is 1:20000, the optimal enzyme-labeled secondary antibody action time is 60min, and the optimal substrate color development time is 10min. Under the above conditions, when S / P=(SN) / (PN)>0.137, it is judged as I-inlH positive, otherwise it is negative.

[0082] As certain embodiments of the present invention, the test samples in step (1) are incubated on ActA coated plates at 37°C for 1 h, and on SmcL and I-inlH coated plates at 37°C for 2.5 h.

[0083] As certain embodiments of the present invention, the test sample in step (1) is serum.

[0084] As certain embodiments of the present invention, in step (2), 100 μl of secondary antibody diluted 1:20,000 is added to the ActA coated plate and SmcL coated plate of step (1) and incubated for 60 min; and 100 μl of secondary antibody diluted 1:10,000 is added to the I-inlH coated plate and incubated for 60 min.

[0085] As certain embodiments of the present invention, in step (3), the color development time of the ActA-coated plate is 5 min, and the color development time of the SmcL and I-inlH-coated plates is 10 min.

[0086] As certain embodiments of the present invention, the detection method further includes a judgment step: when the results of ActA, SmcL and I-inlH detection are all negative, it indicates that the host has not suffered from listeriosis; when the ActA detection result is positive and the SmcL and I-inlH detection 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 4h serotype 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.

[0087] Other test results except for the above cases are invalid and require reference from third-party diagnostic methods.

[0088] 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 detection reagents for detecting Listeria monocytogenes and / or Listeria ivanovii, and the immunoassay reagents also include 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.

[0089] 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.

[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 serotype 4h Listeria monocytogenes or other Listeria monocytogenes.

[0091] The present invention is further described below in conjunction with specific examples, and it should be understood that these examples are only used to illustrate the present invention and are not used 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 details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0092] It should be understood that the terms used in the examples of the present invention are intended to describe specific embodiments rather than to limit the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated, the singular forms "a", "an" and "the" include plural forms. When the examples give numerical ranges, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected.

[0093] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, any methods, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials in the embodiments of the present invention may be used to implement the present invention, based on the prior art mastery of those skilled in the art and the description of the present invention.

[0094] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the technical field and related fields.

[0095] Example 1: Amplification of ActA, SmcL and I-inlH protein encoding genes and construction and expression of expression vectors

[0096] 1. Primer design and synthesis

[0097] Referring to the gene sequences of ActA, SmcL and I-inlH proteins published in GenBank, three pairs of specific primers were designed and synthesized: the upstream primer and downstream primer sequences for amplifying the actA gene are shown in SEQ ID NOs: 7 and 10; the upstream primer and downstream primer sequences for amplifying the smcL gene are shown in SEQ ID NOs: 8 and 11; the upstream primer and downstream primer sequences for amplifying the i-inlH gene are shown in SEQ ID NOs: 9 and 12. Double restriction sites BamH I and HindIII were designed in the upstream and downstream primers, respectively. The primers were synthesized, purified and sequenced by Beijing Qingke Biotechnology Co., Ltd.

[0098]

[0099]

[0100] 2. PCR amplification using the above primers

[0101] Using the Listeria monocytogenes LM4 genome and the Listeria ivanovii XYSL genome as templates, the PCR amplification results are as follows: Figure 1 As shown. The PCR amplification reaction system is: the amplification system is 50μL, including 2× Primestar max DNA Polymerase 25μL, upstream and downstream primers 2μL each, template 2μL, and sterilized ddH2O is added to the final volume of 50μL. The PCR amplification reaction conditions are: 98℃ pre-denaturation for 5min before entering the cycle; 98℃ denaturation for 10s, 55℃ annealing for 15s, 72℃ extension for 1min, 34 cycles; 72℃ final extension for 10min.

[0102] 3. Construction of recombinant plasmid

[0103] 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. The results were as follows Figure 2 The plasmid identified as correct by electrophoresis was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0104] The actA protein sequence is shown in SEQ ID NO: 1:

[0105] ATDSEDSSLNTDEWEEEKTEEQPSEVNTGPRYETAREVSSRDIKELEKSNKVRNTNKADLIAMLKEKAEKGPNINNNNSEQTENAAINEEASGADRPAIQVERRHPGLPSDSAAEIKKRRKAIASSDSELESLTYPDKPTKVNKKKVAKESVADASESDLDSSMQSADESSPQPLKANQQPFFPKVFKKIKDAGKWVRDKIDENPEVKKAIVDKSAGLIDQLLTKKKSEEVNASDFPPPPTDEELRLALPETPMLLGFNAPATSEPSSFEFPPPPTDEELRLALPETPMLLGFNAPATSEPSSFEFPPPPTEDELEIIRETASSLDSSFTRGDLASLRNAINRHSQNFSDFPPIPTEEELNGRGGRPTSEEFSSLNSGDFTDDENSETTEEEIDRLADLRDRGTGKHSRNAGFLPLNPFASSPVPSLSPKVSKISAPALISDITKKTPFKNPSQPLNVFNKKTTTKTVTKKPTPVKTAPKLAELPATKPQETVLRENKTPFIEKQAETNKQSINMPSLPVIQKEATESDKEEMKPQTEEKMVEESESANNANGKNRSA GIEEGKLIAKSAEDEKAKEEPGNHTTLILAMLAIGVFSLGAFIKIIQLRKNN

[0106] The smcL protein sequence is shown in SEQ ID NO:2:

[0107] The SDEYPGNFKITSHNVYLFSRNIYPNWGQMHRADLIAQADYMKNNDVVILNEAFDTSASHRLLNNLREMYPHQTPVIGRSKHGWDKTEGNYSNFALEDGGVAVVSQWPIVEKSQHIFQRGGGADRLSNKGFAYVKIMKNGKPYHIIGTHTQADDSLISKDTSRAIRAEQMQEIQTFIAKKNIPKDEIIFIGGDLNVNYGTDEYHDMLKLLNVSSPANFNGQMATWDPTTNSMLKESYPKAAPEYLDYIFVENGHARPHSWHNKVLHTKSPQWSVKSWFKTYTYQDFSDHYPVVGFTDNNi-inl The protein sequence is shown in SEQ ID NO:3:

[0108] ILHSIPINEIFQDYGLANAVKRQLGKQSVTEVVSQRELDEIQKLNGNGCNIKSIKGVEYLTNLTKLYLSYNHISDISALASLSQLEVLSLKQNKLKDINSLANLTKLRCLYVSNNQLRDLWALEGLKNLEYVDAEEQTCTNNPVVYKPKVVIPNTIKDMSGKSVTPNFISDNGEYINTDVIWELPTYTTEVSYRFKDILNIGKDYVLFSGVVIQPVELEAPAAENAPSINQTLPAASLTEALKKGLRKKAVTDISPKSELDKANEFHDENRGSAMFMQLKKSLLM

[0109] The actA gene sequence is shown in SEQ ID NO:4:

[0110] GCGACAGATAGCGAAGATTCTAGTCTAAACACAGATGAATGGGAAGAAGAAAAAACAGAAGAGCAACCAAGCGAGGTAAATACGGGACCAAGATACGAAACTGCACGTGAAGTAAGTTCACGTGATATTAAAGAACTAGAAAAATCGAATAAAGTGAGAAATACGAACAAAGCAGACCTAATAGCAATGTTGAAAGAAAAAGCAGAAAAAGGTCCAAATATCAATAATAACAACAGTGAACAAACTGAGAATGCGGCTATAAATGAAGAGGCTTCAGGAGCCGACCGACCAGCTATACAAGTGGAGCGTCGTCATCCAGGATTGCCATCGGATAGCGCAGCGGAAATTAAAAAAAGAAGGAAAGCCATAGCATCATCGGATAGTGAGCTTGAAAGCCTTACTTATCCGGATAAACCAACAAAAGTAAATAAGAAAAAAGTGGCGAAAGAGTCAGTTGCGGATGCTTCTGAAAGTGACTTAGATTCTAGCATGCAGTCAGCAGATGAGTCTTCACCACAACCTTTAAAAGCAAACCAACAACCATTTTTCCCTAAAGTATTTAAAAAAATAAAAGATGCGGGGAAATGGGTACGTGATAAAATCGACGAAAATCCTGAAGTAAAGAAAGCGATTGTTGATAAAAGTGCAGGGTTAATTGACCAATTATTAACCAAAAAGAAAAGTGAAGAGGTAAATGCTTCGGACTTCCCGCCACCACCTACGGATGAAGAGTTAAGACTTGCTTTGCCAGAGACACCAATGCTTCTTGGTTTTAATGCTCCTGCTACATCAGAACCGAGCTCATTCGAATTTCCACCACCACCTACGGATGAAGAGTTAAGACTTGCTTTGCCAGAGACGCCAATGCTTCTTGGTTTTAATGCTCCTGCTACATCGGAACCGAGCTCGTTCGAATTTCCACCGCCTCCAACAGAAGATGAACTAGAAATCATCCGGGAAACAGCATCCTCGCTAGATTCTAGTTTTACAAGAGGGGATTTAGCTAGTTTGAGAAATGCTATTAATCGCCATAGTCAAAATTTCTCTGATTTCCCACCAATCCCAACAGAAGAAGAGTTGAACGGGAGAGGCGGTAGACCAACATCTGAAGAATTTAGTTCGCTGAATAGTGGTGATTTTACAGATGACGAAAACAGCGAGACAACAGAAGAAGAAATTGATCGCCTAGCTGATTTAAGAGATAGAGGAACAGGAAAACACTCAAGAAATGCGGGTTTTTTACCATTAAATCCGTTTGCTAGCAGCCCGGTTCCTTCGTTAAGTCCAAAGGTATCGAAAATAAGCGCACCGGCTCTGATAAGTGACATAACTAAAAAAACGCCATTTAAGAATCCATCACAGCCATTAAATGTGTTTAATAAAAAAACTACAACGAAAACAGTGACTAAAAAACCAACCCCTGTAAAGACCGCACCAAAGCTAGCAGAACTTCCTGCCACAAAACCACAAGAAACCGTACTTAGGGAAAATAAAACACCCTTTATAGAAAAACAAGCAGAAACAAACAAGCAGTCAATTAATATGCCGAGCCTACCAGTAATCCAAAAAGAAGCTACAGAGAGCGATAAAGAGGAAATGAAACCACAAACCGAGGAAAAAATGGTAGAGGAAAGCGAATCAGCTAATAACGCAAACGGAAAAAATCGTTCTGCTGGCATTGAAGAAGGAAAACTAATTGCTAAAAGTGCAGAAGACGAAAAAGCGAAGGAAGAACCAGGGAACCATACGACGTTAATTCTTGCAATGTTAGCTATTGGCGTGTTCTCTTTAGGGGCGTTTATCAAAATTATTCAATTAAGAAAAAATAATTAA

[0111] The smcL gene sequence is shown in SEQ ID NO:5:

[0112] AGTGATGAGTATCCAGGTAATTTTAAGATTACTTCACACAACGTTTATTTATTTTCT

[0113] AGAAATATATATCCTAATTGGGGACAAATGCACCGAGCTGATTTAATTGCACAAGCGG

[0114] ACTATATGAAAAATAATGATGTCGTCATTTTAAACGAAGCCTTTGATACAAGTGCTTCA

[0115] CATCGACTATTAAATAACCTTAGAGAAATGTATCCTCATCAAACACCCGTAATTGGGA

[0116] GAAGTAAACATGGTTGGGATAAAACGGAAGGAAATTATTCCAATTTTGCTTTGGAAGA

[0117] TGGTGGAGTGGCTGTCGTTAGCCAGTGGCCTATTGTCGAAAAAAGCCAACATATTTTTC

[0118] AGCGCGGCGGTGGCGCAGATCGGCTCAGTAATAAAGGATTCGCCTATGTTAAAATTAT

[0119] GAAAAACGGAAAACCTTATCATATAATTGGAACTCATACCCAAGCGGATGACTCGCTT

[0120] ATTTCTAAAGATACTAGCCGGGCGATTCGTGCAGAACAAATGCAAGAAATTCAAACAT

[0121] TTATTGCCAAGAAAAATATCCCTAAAGATGAAATTATCTTTATTGGAGGCGATTTGAAC

[0122] GTAAACTACGGTACAGATGAATATCATGATATGTTAAAGCTTTTAAATGTTAGCTCTCC

[0123] GGCAAACTTTAACGGACAGATGGCTACTTGGGATCCAACGACTAATTCGATGTTAAAA

[0124] GAAAGTTATCCTAAAGCAGCACCAGAATACCTTGATTATATTTTTGTTGAAAACGGTCA

[0125] TGCACGTCCACATTCATGGCATAACAAAGTCTTGCATACCAAGTCACCACAGTGGTCTG

[0126] TGAAATCTTGGTTTAAAACTTATACGTATCAAGACTTTTCAGATCATTATCCTGTAGTT

[0127] GGTTTCACTGATAATAACTAA

[0128] The i-inlH gene sequence is shown in SEQ ID NO:5:

[0129] ATTCTACATTCGATACCTATAAATGAAATTTTTCAGGATTATGGTTTAGCGAATGCAGTGAAACGACAATTAGGAAAGCAAAGTGTTACAGAGGTTGTTTCACAAAGAGAACTAGATGAAATACAAAAGTTAAATGGTAATGGTTGCAACATTAAATCTATTAAAGGGGTAGAATATCTTACTAATTTAACAAAGTTATATCTATCTTATAATCATATAAGTGATATTAGCGCGCTTGCTTCTTTGAGTCAACTTGAGGTATTATCTTTAAAGCAGAATAAGCTAAAAGACATCAATTCATTAGCTAACTTGACCAAATTAAGATGTTTGTATGTGAGTAACAATCAATTAAGAGATTTATGGGCATTAGAAGGGCTGAAGAATTTAGAGTATGTAGATGCAGAAGAACAAACCTGCACCAATAATCCTGTTGTATACAAACCAAAAGTGGTTATTCCAAATACGATTAAAGACATGAGCGGAAAGTCAGTTACACCAAATTTTATTAGCGATAATGGAGAATATATAAATACGGATGTTATATGGGAGTTGCCGACATATACAACGGAAGTAAGCTATAGATTTAAGGATATTCTAAACATTGGGAAAGACTACGTTCTATTTAGTGGTGTAGTAATACAGCCAGTGGAATTAGAGGCACCTGCTGCTGAAAATGCACCCTCTATTAACCAAACTTTACCAGCAGCAAGTCTAACAGAAGCACTAAAAAAAGGCTTAAGAAAAAAGGCAGTTACGGATATTAGTCCAAAAAGTGAATTAGATAAGGCGAATGAATTTCACGATGAAAATAGAGGAAGTGCAATGTTTATGCAACTTAAGAAATCTCTACTTATGTGA

[0130] 4. Construction and expression of expression vector

[0131] The positive recombinant plasmid was transformed into BL21(DE3) competent cells, white colonies were picked for purification and identification, and the single colonies BL21(DE3) (pET-30a-actA) identified as positive were inoculated in the presence of Kan + LB (100 μg / mL) liquid medium, shake culture at 37℃ until OD value is 0.4, add IPTG (0.2mM / L), induce at 37℃, 180rpm for 5h, collect bacteria by centrifugation, lyse bacteria by ultrasonic crusher, collect supernatant after centrifugation, purify ActA protein by affinity chromatography column (His-Bind purification kit, purchased from Novagen). rHis-SmcL and rHis-I-inlH sources: except that the induction temperature is 15℃ and the induction time is 20h, the rest of the steps are the same as above. Then SDS-PAGE electrophoresis is performed, and the results are as follows Figure 3 As shown, fusion target protein fragments with molecular weights of 97 kDa, 36 kDa and 35 kDa were obtained.

[0132] Example 2: Western blot and bioreactivity identification of ActA, SmcL and I-inlH recombinant proteins

[0133] 1. Western blot identification of ActA, SmcL and I-inlH proteins

[0134] After the purified recombinant fusion protein was subjected to SDS-PAGE electrophoresis, a Western blot reaction was performed using a His monoclonal antibody solution as the primary antibody and an HRP-labeled goat anti-mouse polyclonal antibody as the secondary antibody. The ECL ultrasensitive luminescent colorimetric solution was used to develop the color in the dark for 2 minutes before reading the slide. The results showed that the three Listeria recombinant proteins of the present invention could react with specific monoclonal antibodies, indicating that the protein identification was correct ( Figure 4 ). The purified and identified recombinant protein was dialyzed and then the protein concentration was determined.

[0135] 2. Bioreactivity identification of ActA, SmcL and I-inlH proteins

[0136] In order 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 identified proteins were loaded into the SDS-PAGE gel wells, and after transfer and sealing, the positive polyclonal antibody-antibody sheep serum infected with Listeria monocytogenes and / or Listeria ivanovii was used as the primary antibody for Western blot reaction, and rabbit anti-sheep enzyme-labeled antibody was used as the secondary antibody for color development. Four different reaction conditions were set up: ① Sheep serum infected with Listeria monocytogenes and Listeria ivanovii; ② Anti-4h Listeria monocytogenes positive sheep serum; ③ Anti-1 / 2a type monocytogenes 4 positive sheep serum; ④ Anti-Listeria ivanovii positive sheep serum. Figure 5 It can be seen that the specific distribution of ActA, SmcL and I-inlH proteins in 4h-type monocytogenes Listeria monocytogenes, non-4h-type monocytogenes Listeria monocytogenes and Listeria ivanovii is shown in Table 1.

[0137] Table 1 Distribution of ActA, SmcL and I-inlH antigens in 4h serotype Listeria monocytogenes, non-4h serotype Listeria monocytogenes and Listeria ivanovii

[0138]

[0139] Note: “+” indicates existence, “-” indicates non-existence.

[0140] Example 3: Establishment of an indirect ELISA method for distinguishing Listeria monocytogenes and Listeria ivanovii from sheep infected with Listeria monocytogenes

[0141] 1. Determination of the optimal antigen coating concentration and enzyme-labeled secondary antibody working concentration

[0142] The optimal coating concentration of the antigen and the optimal dilution multiple of the enzyme-labeled secondary antibody were determined by the ELISA chessboard method. ActA, SmcL and I-inlH proteins were diluted to different concentrations (10μg / mL, 5μg / mL, 2.5μg / mL, 1.25μg / mL, 0.625μg / mL, 0.31μg / mL and 0.16μg / mL) using carbonate buffer, 100μL / well, incubated at 4℃ for 16h, and washed with PBST. The next day, 200μL of PBS containing 2% BSA was added to each well, incubated at 37℃ for 2h, and washed with PBST. The same positive and negative sheep serum used to determine the optimal reaction conditions was diluted 1:200 with PBST solution containing 1% BSA, and the diluted 100.5μL serum sample was added to the enzyme-labeled well, incubated in a 37℃ water bath for 2h, and washed with PBST. Then, the rabbit anti-sheep enzyme-labeled secondary antibody was diluted to 1:5000, 1:10000, 1:20000 and 1:40000 respectively, and 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 OD was read on the ELISA reader after the 2M H2SO4 solution was terminated. 450 Select the positive OD 450 The value is around 1.0 and the negative OD 450 The value is below 0.2 and the OD is positive 450 / Negative OD 450 The condition with a larger (P / N) value was taken as the optimal working concentration of coating antigen and rabbit anti-sheep enzyme-labeled secondary antibody.

[0143] The results are as follows Figure 6 As shown in the figure, considering the cost and dosage of the coated antigen and enzyme-labeled secondary antibody, when the ActA antigen coating concentration is 0.625μg / mL and the enzyme-labeled secondary antibody dilution multiple is 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 multiple is 1:10000, the positive OD 450 The value is closest to 1.0 (1.037); when the I-inlH antigen coating concentration is 1.25μg / mL and the enzyme-labeled secondary antibody dilution multiple is 1:20000, the positive OD 450The value is closest to 1.0 (1.081). At this time, the indirect ELISA reaction is the most stable and the P / N value is large under this condition. Therefore, the optimal antigen coating concentration and the optimal enzyme-labeled secondary antibody dilution ratio of indirect ELISA were determined to be 0.625μg / mL, 1:40000 (ActA), 0.625μg / mL, 1:10000 (SmcL) and 1.25μg / mL, 1:20000 (I-inlH), and the secondary antibody concentrations at this time were 50ng / mL, 200ng / mL and 100ng / mL, respectively.

[0144] 2. Determination of coating conditions

[0145] Based on the optimal reaction conditions determined above, 4 groups of coating conditions were set up and the OD of each group was compared. 450 and P / N value, select the coating condition with less time cost and the largest P / N value as the optimal coating condition. Figure 7 It can be seen that the optimal coating condition for the ActA indirect ELISA method is 37°C for 2 h, and the optimal coating condition for the SmcL and I-inlH indirect ELISA methods is 4°C for 16 h.

[0146] 3. Determination of blocking solution

[0147] Based on the optimal reaction conditions determined above, 4 groups of blocking solution conditions were set respectively, and the blocking solution corresponding to the maximum P / N value was selected as the optimal blocking solution. Figure 8 As shown, the ActA indirect ELISA method selected PBS containing 5% BSA as the optimal blocking solution; the SmcL and I-inlH indirect ELISA methods selected PBS containing 5% skim milk as the optimal blocking solution.

[0148] 4. Determination of closure time

[0149] Based on the optimal reaction conditions determined above, four different blocking times were set, and the condition with the largest P / N average value was selected as the optimal blocking time. Fig. 9 As shown, the ActA indirect ELISA method selected 2.5h as the optimal blocking time; the SmcL and I-inlH indirect ELISA methods selected 1.5h as the optimal blocking time.

[0150] 5. Determination of serum primary antibody incubation time

[0151] Based on the optimal reaction conditions determined above, the positive and negative control sera were diluted at a serum dilution multiple of 1:200 and reacted at 37°C for 1h, 1.5h, 2h and 2.5h, respectively. Fig.10As shown, the indirect ELISA method for ActA selected an incubation time of 1 h as the optimal primary antibody incubation time; the indirect ELISA method for SmcL and I-inlH selected an incubation time of 2.5 h as the optimal primary antibody incubation time.

[0152] 6. Determination of enzyme-labeled antibody reaction time

[0153] Based on the optimal reaction conditions determined above, different HRP-rabbit anti-goat IgG enzyme-labeled antibody incubation times were set to 30 min, 45 min, 60 min and 75 min. Fig.11 As shown, the ActA indirect ELISA method selected 30 min as the optimal secondary antibody reaction time; the SmcL and I-inlH indirect ELISA methods selected 60 min as the optimal secondary antibody reaction time.

[0154] 7. Determination of substrate color development time

[0155] Based on the optimal reaction conditions determined above, four different TMB color development times were set. Fig.12 As shown, the ActA indirect ELISA method selected 5 minutes as the optimal color development time; the SmcL and I-inlH indirect ELISA methods selected 10 minutes as the optimal color development time.

[0156] Example 4: Assembly of Sheep Listeriosis ELISA Kit and Determination of Threshold

[0157] The ELISA kit consists of: 3 antigen-coated plates (1 each of the ELISA plates coated with ActA, SmcL and I-inlH proteins), 800 μL each of the positive control serum and negative control serum diluted at 1:200, 50 mL of antibody diluent (1x), 1 mL of rabbit anti-sheep ELISA antibody diluted at 1:1000 (diluted with antibody diluent to the optimal ratio for each method when used), 60 mL of PBST washing solution (20×), 30 mL of TMB substrate colorimetric solution, 20 mL of 2 mol / L H2SO4 stop solution and 1 instruction manual.

[0158] The threshold of the ELISA method with ActA, SmcL and I-inlH as the coating antigen was determined according to the ELISA reaction conditions determined in Example 3. The specific operation was to test 94 negative sera tested against Listeria monocytogenes by a third-party kit, and the positive and negative control test values ​​were recorded as P and N. The average value of the 94 negative serum test results was calculated. and standard deviation SD, to obtain the critical value Cut-off value = (LN) / (PN), and the sample is tested at OD 450The value is recorded as S. If S / P=(SN) / (PN)>Cut-off value, it is judged as positive serum, otherwise it is negative serum.

[0159] The results are shown in Table 2(a), (b) and (c). The average values ​​of the ActA-iELISA method are The cut-off value was calculated to be 0.104. The average value of the SmcL-iELISA method was The cut-off value was calculated to be 0.110. The average value of the I-inlH-iELISA method was 0.179, the standard deviation SD was 0.018, and the cut-off value was calculated to be 0.110. The cut-off value is 0.137.

[0160] Table 2 (a) ActA indirect ELISA threshold determination results

[0161] ActA 1 2 3 4 5 6 7 8 9 10 11 12 A 0.14 0.143 0.128 0.23 0.148 0.135 0.148 0.148 0.146 0.192 0.14 0.15 B 0.132 0.137 0.224 0.135 0.15 0.135 0.227 0.222 0.14 0.137 0.135 0.135 C 0.139 0.137 0.129 0.229 0.145 0.157 0.136 0.234 0.163 0.159 0.146 0.143 D 0.15 0.141 0.139 0.228 0.153 0.151 0.145 0.144 0.233 0.156 0.14 0.152 E 0.138 0.145 0.138 0.143 0.148 0.153 0.141 0.151 0.151 0.151 0.156 0.165 F 0.156 0.162 0.148 0.154 0.163 0.159 0.149 0.151 0.141 0.154 0.154 0.16 G 0.146 0.159 0.153 0.142 0.163 0.156 0.151 0.155 0.156 0.171 0.173 0.968(P) H 0.163 0.171 0.153 0.155 0.18 0.168 0.151 0.156 0.159 0.168 0.161 0.143(N)

[0162] Table 2(b) SmcL indirect ELISA threshold determination results

[0163]

[0164]

[0165] Table 2 (c) I-inlH indirect ELISA threshold determination results

[0166] I-inlH 1 2 3 4 5 6 7 8 9 10 11 12 A 0.17 0.172 0.168 0.189 0.17 0.172 0.169 0.17 0.173 0.174 0.179 0.193 B 0.191 0.17 0.175 0.169 0.178 0.184 0.168 0.193 0.187 0.167 0.171 0.171 C 0.172 0.17 0.16 0.173 0.179 0.178 0.167 0.167 0.169 0.175 0.171 0.19 D 0.179 0.178 0.174 0.179 0.209 0.186 0.175 0.183 0.185 0.172 0.195 0.198 E 0.193 0.191 0.183 0.188 0.19 0.181 0.172 0.175 0.19 0.186 0.194 0.158 F 0.187 0.178 0.183 0.176 0.181 0.184 0.176 0.184 0.183 0.185 0.184 0.182 G 0.19 0.183 0.178 0.273 0.178 0.186 0.187 0.18 0.187 0.187 0.194 0.866(P) H 0.215 0.187 0.228 0.191 0.194 0.193 0.194 0.159 0.2 0.188 0.191 0.129(N)

[0167] Example 5: Repeatability test of sheep listeriosis ELISA kit

[0168] Repeatability test was performed according to the indirect ELISA method established in Example 3. The intra-batch repeat stability test was to use the same batch of purified protein coated ELISA plate to test the same 6 sheep sera for 3 consecutive days; the inter-batch repeat stability test was to use 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.

[0169] The results are shown in Table 3 (a), (b) and (c). The intra-batch CV of the ActA-iELISA method is between 2.45% and 6.74%, and the inter-batch CV is between 3.15% and 8.28%; the intra-batch CV of the SmcL-iELISA method is between 1.42% and 5.54%, and the inter-batch CV is between 5.25% and 9.61%; the intra-batch CV of the I-inlH-iELISA method is between 1.96% and 7.23%, and the inter-batch CV is between 4.46% and 12.48%. The above results show that the ELISA detection kit has a small degree of variation and good repeatability.

[0170] Table 3 (a) ActA-iELISA repeatability test results

[0171]

[0172] Table 3 (b) SmcL-iELISA repeatability test results

[0173]

[0174]

[0175] Table 3 (c) I-inlH-iELISA repeatability test results

[0176]

[0177] Example 6: Practicality test of sheep listeriosis ELISA kit

[0178] 1. Dynamic changes of ActA and SmcL antibody levels

[0179] The 4h serotype of Listeria monocytogenes strain XYSN was cultured 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 serum, and the production pattern of ActA and SmcL antibodies in sheep serum was analyzed. The production time of ActA and SmcL antibodies in Hu sheep after infection with XYSN was as follows Fig.13 As shown in A and 13B, all Hu sheep were tested positive by the ActA-iELISA method 6 days after infection, that is, they were infected with listeriosis. The antibody levels mostly reached the peak around the 18th day, and the 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.

[0180] 2. Dynamic changes of I-inlH antibody levels

[0181] Listeria monocytogenes strain XYSL was cultured at 3×10 9 6-week-old BALB / c mice were orally infected with a dose of CFU. The I-in1H antibody in the serum was detected according to the indirect ELISA method established in Example 3. The results are shown in FIG. Fig.13 As shown in C, BALB / c mice were tested positive for listeriosis 6 days after infection, with the highest antibody level on day 16, and antibodies against I-inlH were still at a high level 30 days after infection. The results show that the I-inlH detection component in the kit can diagnose listeriosis in the early stage of host infection and for a long time after infection.

[0182] Example 7: Thermal stability of ELISA test kit for sheep listeriosis

[0183] The heat destruction operation of the antigen coated plate is to coat and seal according to the optimal conditions determined in Example 3, then put it in an oven for drying, put it in aluminum foil for vacuum packaging and place it in a 37°C constant temperature incubator for 6, 7 and 8 days. The heat destruction operation of the rabbit anti-sheep enzyme-labeled antibody is to use Candor's HRP-ProtectorTM protective agent to dilute it at 1:50 and seal it, then place it in a 37°C constant temperature incubator for 6, 7 and 8 days. The heat destruction operation of the serum control is to place the diluted positive and negative control sera in a 37°C constant temperature incubator for 6, 7 and 8 days. The indirect ELISA method established in Example 3 was used to detect the intra-batch coefficient of variation of the three control sera. Among them, Positive reference is a positive reference, and Negative reference is a negative reference. The test results are shown in Tables 4 (a), (b) and (c). It is found that the intra-batch coefficient of variation of all components in the kit under heat destruction conditions is less than 15%, indicating that the thermal stability of the entire kit meets the standard, and its shelf life at 4°C is predicted to be more than 8 months.

[0184] Table 4 (a) Thermal stability analysis of ActA kit

[0185]

[0186] Table 4 (b) Thermal stability analysis of SmcL kit

[0187]

[0188] Table 4 (c) Thermal stability analysis of I-inlH kit

[0189]

[0190] Example 8: Specificity and sensitivity of the sheep listeriosis ELISA kit

[0191] According to the indirect ELISA method established in Example 3, the specificity and sensitivity of the three detection components in the detection kit are calculated as shown in Tables 5 (a), (b) and (c). The ActA detection kit was used to detect 65 sheep sera infected with Listeria monocytogenes and 92 uninfected sera, and 61 positive samples were detected, so the sensitivity was 93.85%; 87 negative samples were detected, and the specificity was 94.57%. The SmcL detection kit was used to detect 70 sheep sera infected with 4h-type Listeria monocytogenes or Listeria ivanovii and 92 uninfected sera, and 66 positive samples were detected, so the sensitivity was 94.29%; 88 negative samples were detected, and the specificity was 95.65%. The I-inlH detection kit was used to detect 15 sheep sera infected with Listeria ivanovii and 92 uninfected sera, and 15 positive samples were detected, so the sensitivity was 100%; 87 negative samples were detected, so the specificity was 94.57%. The source of all sera is clear and tested by third-party kits. The results show that the detection components in the ELISA kit have good sensitivity and specificity.

[0192] Table 5 (a) Sensitivity and specificity analysis of ActA kit

[0193] Infectious serum Uninfected serum ActA test positive 61 5 ActA test kit negative 4 87 total 65 92 Sensitivity (%) 93.85％ / Specificity (%) / 94.57％

[0194] Table 5(b) Sensitivity and specificity analysis of SmcL kit

[0195] Infectious serum Uninfected serum SmcL test positive 66 4 SmcL test negative 4 88 total 70 92 Sensitivity (%) 94.29％ / Specificity (%) / 95.65％

[0196] Table 5 (c) Analysis of sensitivity and specificity of I-inlH kit

[0197] Infectious serum Uninfected serum I-inlH test kit positive 15 5 I-inlH test kit negative 0 87 total 15 92 Sensitivity (%) 100％ / Specificity (%) / 94.57％

[0198] Example 9: Compliance test of sheep listeriosis ELISA kit

[0199] The sheep listeriosis ELISA kit described in the present invention was used to test several clinical sheep sera with clear backgrounds stored in the laboratory in the early stage, and the test results were compared with the test results 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 a total of 75 samples had consistent test results, with a total compliance rate of 94.94%; for the SmcL kit, a total of 79 clinical sheep serum samples were tested, and a total of 72 samples had consistent test results, with a total compliance rate of 91.14%; for the I-inlH kit, a total of 50 clinical sheep serum samples were tested, and a total of 46 samples had consistent test results, with a total compliance rate of 92%.

[0200] Table 6 (a) Comparison of ActA kit and Diatheva kit

[0201]

[0202] Table 6 (b) Comparison of SmcL kit and Diatheva kit

[0203]

[0204] Table 6 (c) Comparison of I-inlH kit and Diatheva kit

[0205]

[0206] Example 10: Application of ELISA Detection Kit for Sheep Listeriosis

[0207] The indirect ELISA detection kit of the present invention was used to trace the source of the sheep in the sheep farm of Example 10, and a total of 40 sheep serum samples were collected from a domestic sheep farm for testing. The results showed that 3 sheep were positive for ActA and SmcL antibodies, that is, they were infected with 4h serotype monocytogenes Listeria; 1 sheep was positive for ActA antibodies, that is, it was infected with non-4h serotype monocytogenes Listeria; 12 sheep were positive for SmcL and I-inlH antibodies, that is, they were infected with Listeria ivanovii; 15 sheep were positive for ActA, SmcL and I-inlH antibodies, that is, they were mixedly infected with Listeria monocytogenes + Listeria ivanovii, and the overall antibody positive 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 relatively high, and prevention, control and disinfection should be carried out well.

[0208] Table 7 ELISA kit for sheep listeriosis detection of sheep serum samples from a certain place in East China

[0209] Types of infection A B C D E total 4h Lm 2 0 0 0 1 3 Non-4h Lm 1 0 0 0 0 1 Liv 1 2 2 5 2 12 Lm+Liv 2 4 5 2 2 15 Number of positive 6 6 7 7 5 31 sum 8 8 8 8 8 40 Positive rate 75％ 75％ 87.5％ 87.5％ 62.5％ 77.5％

[0210] The inventors used bioinformatics software to predict the hydrophilicity, surface polarity, antigenicity and secondary structural characteristics of the I-inlH protein in the early stage, and predicted and analyzed the antigenic epitope regions that may exist in the protein. The inventors found that the truncated fragments of the protein have good conservatism and immunogenicity. After constructing and expressing ActA, SmcL and I-inlH proteins successfully, the specific distribution of these three antigens in 4h serotype monocytogenes Listeria, other serotype monocytogenes Listeria and Listeria ivanovii was verified. Based on this premise, the inventors studied and designed an indirect ELISA kit for distinguishing between Listeria monocytogenes and Listeria ivanovii species infections of Listeria monocytogenes of sheep. The kit of the present invention can effectively distinguish sheep infected with 4h serotype monocytogenes Listeria monocytogenes, non-4h serotype monocytogenes Listeria monocytogenes, Listeria ivanovii or mixed infection of Listeria monocytogenes and Listeria ivanovii, and has good specificity, sensitivity and repeatability, which is convenient for large-scale detection of listeriosis in sheep farms.

[0211] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. 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, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes 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 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 OD450 nm value - negative control OD450 nm average value) / (positive control OD450 nm average value - negative control OD450 nm 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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