Detection method and kit for detecting listeria monocytogenes

Abstract

The invention discloses a detection method and a kit for detecting listeria monocytogenes, and relates to the technical field of biology. The kit is an indirect ELISA kit and comprises an elisa plate coated with an antigen, the amino acid sequence of the antigen is as shown in SEQ ID NO. 2. The indirect ELISA detection kit constructed by the invention only reacts with listeria monocytogenes positive serum, has no cross reaction with other serum, and has strong specificity; when the serum dilution is 1: 800, the detection result is still positive, and the sensitivity is high; the intra-batch variation coefficient ranges from 2.79% to 6.64%, the inter-batch variation coefficient ranges from 2.61% to 7.75%, and the intra-batch repeatability and the inter-batch repeatability are good; 24 serum samples are detected, and the total coincidence rate is 95.83%. The kit provided by the invention can effectively detect sample infection of Listeria monocytogenes, and has the advantages of strong specificity, high sensitivity and good repeatability.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a detection method and a kit for detecting Listeria monocytogenes. Background Art

[0002] Listeria monocytogenes (Listeria monocytogenes), also known as L. monocytogenes, is a Gram-positive intracellular parasitic pathogen that can be transmitted to humans and animals. It is widely found in nature and is primarily transmitted through the fecal-oral route via contaminated food (such as unpasteurized dairy products, cooked meats, and ready-to-eat fruits and vegetables). It can also be transmitted through damaged skin, mucous membranes, or the placenta. Humans, cattle, sheep, and pigs can all be infected with Listeria. Pregnant women, newborns, the elderly, and those with immunodeficiency are susceptible to infection. Infection in pregnant women can lead to miscarriage, stillbirth, or neonatal sepsis and meningitis. Immunocompromised individuals are more likely to develop sepsis and meningitis.

[0003] The virulence factors of Listeria monocytogenes mainly include hemolytic toxins, actin polymerases, C-type phospholipases, internalizing proteins, cell wall hydrolases, metalloproteinases, amidases and regulatory factors. After Listeria monocytogenes invades the host, it can cause multiple reactions in the host cells and cause the host to produce an immune response, thereby producing antibodies to resist the invasion of pathogens. Therefore, serum testing is an important basis for diagnosing the disease. Among them, internalizing proteins can increase the ability of Listeria to invade host cells, enhance the virulence of Listeria, and become an important target for vaccine development and diagnosis due to its good immunogenicity. The present invention intends to optimize the expression conditions to achieve soluble expression of the InlB protein, ensure its natural conformation, and use the InlB recombinant protein to establish an indirect ELISA method for detecting listeriosis, thereby providing a reference basis for the monitoring and diagnosis of listeriosis. Summary of the Invention

[0004] The present invention aims to provide a method and kit for detecting Listeria monocytogenes to address the problems of the prior art. The kit can effectively detect samples infected with Listeria monocytogenes and has the advantages of high specificity, high sensitivity, and good reproducibility.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an indirect ELISA kit for detecting antibodies to Listeria monocytogenes, comprising an ELISA plate coated with an antigen;

[0007] The antigen is a recombinant InlB protein, and its amino acid sequence is shown in SEQ ID NO.2.

[0008] Furthermore, the preparation method of the recombinant InlB protein comprises the following steps:

[0009] The inlB gene was homologously recombined with the PColdⅡ-10His plasmid to obtain the recombinant plasmid PColdⅡ-10His-inlB;

[0010] The recombinant plasmid PColdⅡ-10His-inlB was transformed into competent Escherichia coli cells to obtain a recombinant expression strain;

[0011] After inducing expression culture of the recombinant expression strain, the recombinant InlB protein is obtained by protein purification;

[0012] The nucleotide sequence of the inlB gene is shown in SEQ ID NO.1.

[0013] Furthermore, the method for preparing the antigen-coated ELISA plate comprises the following steps:

[0014] The antigen is diluted with a coating solution to obtain a suspension, and the suspension is added to an enzyme-labeled well for coating treatment. A blocking solution is then added for blocking treatment to obtain the antigen-coated enzyme-labeled plate.

[0015] Furthermore, the concentration of the recombinant InlB protein in the suspension is 4 μg / mL.

[0016] Furthermore, the blocking solution is 10% skim milk powder.

[0017] Furthermore, the indirect ELISA kit also includes an enzyme-labeled secondary antibody, a diluent, a washing solution, a color developing solution, a stop solution, a negative standard and a positive standard.

[0018] Furthermore, the diluent is PBST buffer; and / or

[0019] The washing solution is PBS buffer containing 0.1% Tween 20.

[0020] Furthermore, the color developing solution is TMB solution.

[0021] Furthermore, the stop solution is 2 mol / L H2SO4 solution.

[0022] The present invention also provides a method for detecting antibodies to Listeria monocytogenes for non-disease diagnosis purposes, comprising the steps of detecting antibodies to Listeria monocytogenes using the above-mentioned indirect ELISA kit:

[0023] Add negative control, positive control and sample solution to the antigen-coated ELISA plate respectively for the first incubation reaction;

[0024] Enzyme-labeled secondary antibody was added to each reaction well for a second incubation reaction;

[0025] Add color development solution to each reaction well and continue incubation;

[0026] Add stop solution to each reaction well, measure the absorbance value at a wavelength of 450nm using an enzyme reader, and judge the result.

[0027] The present invention discloses the following technical effects:

[0028] The present invention utilizes the PColdⅡ-10His plasmid to solublely express the InlB recombinant protein in vitro. Soluble protein expression was detected by SDS-PAGE and Western blotting, and the protein was purified using His Ni NTA beads. Furthermore, by optimizing the coating volume and blocking solution, an indirect ELISA assay for listeriosis was established, and its specificity, sensitivity, and reproducibility were tested. Results showed that after overnight induction with 0.5 mM IPTG at 16°C and 150 rpm for 16 hours, Western blotting revealed soluble expression of the InlB recombinant protein. After optimization, it was found that when the InlB recombinant protein was coated at 4 μg / mL, blocked with 10% skim milk powder at 37°C for 1 hour, the test serum was diluted 1:400 and incubated at 37°C for 1 hour, the enzyme-labeled secondary antibody was diluted 1:2500 and incubated at 37°C for 30 minutes, and the color was developed for 8 minutes, the positive / negative cutoff value was 0.37, indicating that an indirect ELISA assay for listeriosis could be established. The present invention also found that the established indirect ELISA detection kit only reacts with Listeria monocytogenes-positive serum and has no cross-reaction with other sera, with strong specificity; when the serum dilution is 1:800, the test result is still positive, with high sensitivity; the intra-batch coefficient of variation is between 2.79% and 6.64%, and the inter-batch coefficient of variation is between 2.61% and 7.75%, with good intra-batch and inter-batch reproducibility; 24 serum samples were tested, and the total compliance rate was 95.83%.

[0029] The indirect ELISA method established by the present invention can have good reactivity with listeriosis positive serum, and the method has strong specificity, high sensitivity and good repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1 The identification results of the PCR amplification products of the inlB gene; where M is the DNA relative molecular weight standard; 1 is the amplified fragment of the inlB gene; 2 is the negative control;

[0032] Figure 2 The PCR identification results of the recombinant plasmid PcoldⅡ-10His-inlB were obtained. M: DNA Marker; 1 to 5: PcoldⅡ-10His-inlB plasmids; 6: negative control.

[0033] Figure 3 The SDS-PAGE analysis results of the induced expression of IlnB recombinant protein; M: Protein Marker; 1: supernatant after induction; 2: precipitate after induction; 3: uninduced bacterial solution; 4: supernatant after induction with pColdⅡ-10His empty vector; 5: precipitate after induction with pColdⅡ-10His empty vector;

[0034] Figure 4 The results of Western blot analysis of purified InlB recombinant protein are shown in Figure 2. M: Protein Marker; 1: Purified InlB recombinant protein. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] Example 1 Establishment and Optimization of Indirect ELISA Detection Method for Listeria monocytogenes

[0041] 1 Materials and Methods

[0042] 1.1 Materials

[0043] 1.1.1 Strains, serum, and vectors

[0044] The inactivated Listeria monocytogenes solution, positive quality control products, and negative quality control products were provided by Beijing Agricultural College; the mouse brucellosis-positive serum, mouse bovine tuberculosis-positive serum, mouse Clostridium perfringens-positive serum, and pColdII-10His plasmid were all preserved by the public health and biosafety control team of the Institute of Animal Husbandry and Veterinary Medicine, Beijing Institute of Animal Husbandry and Veterinary Medicine.

[0045] 1.1.2 Main reagents and instruments

[0046] Goat Anti-Mouse IgG (H+L), HRP Conjugate (Beijing Quanshijin Biotechnology Co., Ltd.); Ni NTA beads (Changzhou Tiandirenhe Biotechnology Co., Ltd.); PrimeSTAR HSDNA Polymerase (Baoriyi Biotechnology (Beijing) Co., Ltd.); 2× Rapid Taq Master Mix, ClonExpress MultiS One Step Cloning Kit (Nanjing Novozymes Biotechnology Co., Ltd.); IPTG, gel preparation kit, page gel staining solution, BSA (Beijing Solebao Company); dialysis bag, primary antibody diluent, His-tag primary antibody, HRP-labeled secondary antibody (Beyotime Biotechnology); Blue IV Protein Marker (Beijing Quanshijin Biotechnology Co., Ltd.); StarMarker D2000 Plus (Kangrun Bio-Genestar); skim milk powder (BD); restriction endonuclease, exposure solution, TMB color development solution, 96-well detachable ELISA plate (ThermoFisher Scientific).

[0047] PCR amplifier T100 Thermal Cycler (Bio-Rad), NanoDrop One (ThermoFisherScientific), nucleic acid electrophoresis instrument, vertical electrophoresis instrument, electroblotting instrument (BioRad), exposure instrument (Tecan), spectrophotometer (ThermoFisher Scientific), four-dimensional rotating mixer BE-1100 (Haimen Qilin Bell Instrument Manufacturing Co., Ltd.), ultrasonic cell disruptor SCIENTZ-IID (Ningbo Xinzhi Biotechnology Co., Ltd.), etc. were all provided by the animal biosafety and public health prevention and control team of the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0048] 1.1.3 Gene and protein sequences

[0049] The nucleotide sequence of the Listeria monocytogenes inlB gene is shown in SEQ ID NO.1, and the amino acid sequence of the Listeria monocytogenes InlB protein is shown in SEQ ID NO.2.

[0050] SEQ ID NO.1:

[0051]

[0052] SEQ ID NO.2:

[0053] .

[0054] 1.2 Methods

[0055] 1.2.1 Primer design

[0056] Primer design was based on the L. monocytogenes EGD gene sequence (HG421741) included in NCBI. SnapGene was used to design the full-length inlB gene amplification primers: inlB-F and inlB-R, which were synthesized by Beijing Qingke Biotechnology Co., Ltd. The specific sequences are shown in Table 1.

[0057] Table 1 Primer sequences for amplifying the inlB gene

[0058]

[0059] 1.2.2 Construction of prokaryotic expression plasmid for InlB protein

[0060] Using an inactivated Listeria monocytogenes culture as a template, the inlB gene fragment was amplified using the primer pair inlB-F / R. A 50 μL PCR reaction system consisted of 25 μL PrimeSTAR Max DNA Polymerase, 20 μL sterile enzyme-free water, 2 μL of each primer, and 1 μL of template. The mixture was then centrifuged. The PCR amplification procedure included initial denaturation at 98°C for 2 minutes, denaturation at 98°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 90 seconds. The reaction was repeated 35 times, followed by a final extension at 72°C for 5 minutes and storage at 4°C.

[0061] The pColdII-10His empty vector plasmid was double-digested with Kpn I and Hind III. The total digestion system (50 μL) included 5 μL of 10× rCutsmart, 1 μg of the pColdII-10His empty vector plasmid, and 1 μL each of Kpn I and Hind III endonucleases. Add ddH2O to a total volume of 50 μL. Mix the components thoroughly, centrifuge, and incubate the digestion reaction in a 37°C water bath for 2 hours. The inlB gene amplification product and digestion products were then analyzed by agarose gel electrophoresis. The correctly sized band was excised and DNA was recovered from the agarose gel.

[0062] The recovered inlB gene fragment was ligated with the linearized pColdII-10His empty vector. The ligation system was 20 μL, including 4 μL of 5×CE MultiS Buffer, 2 μL of Exnase MultiS, 0.03 pmol of linearized vector and inlB gene fragment respectively. ddH2O was added to make the total volume 20 μL. The above components were mixed and centrifuged. The mixture was placed in a 37°C water bath for reaction for 30 minutes. After the reaction, it was immediately cooled on ice.

[0063] Then, 10 μL of the above ligation product was transformed into competent E. coli, and a single clone was picked for bacterial culture. After PCR identification using a primer pair (M13-F / R, see Table 2), the positive bacterial sample was sequenced, and the plasmid was extracted from the bacterial sample with correct sequencing. The extracted plasmid was named pColdII-10His-inlB, and the concentration was detected and stored at -20°C.

[0064] Table 2 Primer sequences for identifying pColdII-10His vector

[0065]

[0066] 1.2.3 Induced expression of recombinant positive bacteria

[0067] The positive recombinant plasmid PColdⅡ-10His-inlB was transformed into Escherichia coli BL21 competent cells to obtain a recombinant expression strain; the recombinant expression strain was inoculated into 4 mL 2×YT liquid medium containing ampicillin (100 mg / mL), and the expression was induced by transferring the fresh bacterial liquid to 20 mL 2×YT liquid medium containing ampicillin (100 mg / mL) at a transfer concentration of 1:100. The OD was measured. 600 The value was 0.6-0.8, IPTG was added with a final concentration of 0.5 mM, and the induction conditions were 16 h, 150 rpm, and 16 °C.

[0068] 1.2.4 Protein immunoblotting

[0069] The bacterial solution was centrifuged at 4400rpm for 8min, the supernatant was discarded, and the solution was washed once with 2mL PBS. After centrifugation at the same speed, the supernatant was discarded, and 1mL PBS was added to reselect the bacterial solution. The solution was transferred to a 2mL Ep tube for ultrasonic disruption and centrifuged at 12000rpm for 20min at 4°C. The supernatant and precipitate were collected separately and identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and protein immunoblotting (Western blot, WB). The identification conditions were: 300mA, transfer for 1.5h, 5% skim milk powder was blocked on a shaker at room temperature for 2h, skim milk was rinsed with PBST, His tag primary antibody (diluted with primary antibody diluent 1:5000), incubated on a shaker at room temperature for 2h (or incubated overnight at 4°C), the membrane was washed 3 times on a shaker with PBST for 5min each time, and then the PBST was discarded and added. Goat Anti-Mouse IgG (H+L), HRP secondary antibody (1:5000 dilution with 5% skim milk powder), incubate on a shaker at room temperature for 1 h, repeat the above membrane washing operation, and finally expose.

[0070] 1.2.5 Purification of InlB protein

[0071] Repeat the steps in 1.2.3 to induce expression. After His Ni NTA beads were treated with Lysis Buffer (10 mM imidazole) and ultrapure water in advance, the supernatant containing the expressed protein was hung on His Ni NTA beads and incubated on a four-dimensional rotator for 4 hours. The flow-through was dripped down the gravity column and washed with self-prepared Washbuffer (20 mM imidazole) until the OD was measured. 280 The value was lower than 0.01, and then the protein was eluted with Elushin buffer (250 mM imidazole) and the OD 280If the value falls below 0.2, stop elution. Take 80 μL of the eluted protein sample for PAGE analysis. The remaining protein proceeds to the next step of dialysis. Replace the imidazole with PBS, changing the PBS regularly for four times before collecting the protein. Only proceed to the next step of the experimental process after confirming that the bands in the eluted sample are of accurate size, single, and meet the experimental requirements.

[0072] 1.2.6 Optimization of indirect ELISA conditions

[0073] Indirect ELISA method:

[0074] (1) diluting the serum sample with a diluent and mixing thoroughly to obtain a sample solution;

[0075] (2) Add 100 μL of diluent, negative control, positive control, and sample solution to the enzyme-labeled wells, respectively, as blank group, negative control group, positive control group, and experimental group, and perform duplicate well experiments, and then incubate at 37°C;

[0076] Negative quality control: negative quality control serum containing antibodies that are not infected with Listeria monocytogenes;

[0077] Positive quality control product: Listeria monocytogenes InlB protein antibody positive quality control serum.

[0078] (3) Discard the unbound sample solution and rinse three times with 200 μL of washing solution; the washing solution is PBST buffer;

[0079] (4) Dilute the enzyme-labeled secondary antibody (Goat Anti-Mouse IgG (H+L), HRP Conjugate) with diluent, add 100 μL of the diluted enzyme-labeled secondary antibody to each reaction well, and repeat step (3) for washing;

[0080] (5) Add 200 μL of TMB substrate solution to each reaction well and incubate at 37°C in the dark;

[0081] (6) Add 50 μL of stop solution (2 mol / L H2SO4 solution) to each reaction well. After incubation, measure the absorbance of each reaction well at a wavelength of 450 nm using a microplate reader.

[0082] (7) Result determination: The positive and negative quality control products and 30 Pasteurella multocida negative sera were tested and the OD was measured using an enzyme marker. 450 The values were calculated, the mean (X) and standard deviation (SD) were calculated, and the positive critical value (X+3×SD) was calculated. When the S / P value of the test sample was greater than X+3×SD, it was judged to be positive, otherwise it was negative.

[0083] Optimize different conditions in the above detection method:

[0084] Determine the optimal coating concentration of InlB protein. Using coating solution (0.293 g sodium bicarbonate, 0.159 g sodium carbonate, add ddH2O to 100 mL, adjust pH to 7.4), dilute the purified InlB protein to 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.25 μg / mL, and 0.125 μg / mL suspensions. Add 100 μL of this suspension to each well and coat overnight at 4°C. The next day, wash the plates five times with PBST (2 L PBS phosphate buffered saline plus 2 mL Tween-20) and block with 5% skim milk powder at 37°C for 1 hour. Add the primary antibody (1:400 dilution of negative and positive serum in PBST) and incubate at 37°C for 1 hour. After washing, add enzyme-labeled secondary antibody Goat Anti-Mouse IgG (H+L), HRP Conjugate (1:2500 diluted in PBST) and incubate at 37°C for 30 minutes. After washing, develop color (TMB single-component colorimetric solution) for 10 minutes under dark conditions, add 50 μL of stop solution, and detect OD 450 value.

[0085] The optimal blocking solution concentration of InlB protein was determined. ELISA plates were coated with InlB protein at a concentration of 1 μg / mL and blocked with 5% skim milk, 10% skim milk, and 10% BSA, respectively. ELISA tests were performed under the same conditions and the OD values were measured. 450 The optimal blocking solution concentration was selected.

[0086] 1.2.7 Specificity Detection

[0087] According to the indirect ELISA method established in 1.2.6, test the Brucella-positive mouse serum, Mycobacterium tuberculosis-positive mouse serum, Clostridium perfringens epsilon protein-positive mouse serum, and Listeria monocytogenes-positive mouse serum respectively, and set up duplicate wells to observe whether the InlB protein cross-reacts with other pathogens to determine its specificity.

[0088] 1.2.8 Sensitivity testing

[0089] The positive control product was diluted to different concentrations, namely 1:800, 1:400, 1:200, 1:100, and 1:50, and the indirect ELISA method established in 1.2.6 was used for detection. At the same time, a negative control was set up, and 3 replicates were set up for each gradient. By comparing the P / N value (the average OD value of the positive control product), the serum dilution was 1:800, 1:400, 1:200, 1:100, and 1:50, respectively. 450 Value / average OD of negative control 450 value) to judge the sensitivity of the method.

[0090] 1.2.9 Repeatability test

[0091] Repeatability test within the batch: Coat a batch of enzyme-labeled plates according to the indirect ELISA method established in 1.2.6, block and wash the next day, then add 90 μL of 50% glycerol to each well, incubate at room temperature for 10 minutes, shake off the glycerol and store in a vacuum at 4°C. Select Listeria monocytogenes positive and negative quality control products, select a preserved enzyme-labeled plate on the 1st, 3rd and 5th day respectively to test the above serum, and set up duplicate wells. After the test is completed, use a microplate reader to read the OD of each well. 450 The intra-assay coefficient of variation was calculated according to the formula: coefficient of variation (CV) = (SD ÷ X) × 100%.

[0092] Inter-batch reproducibility test: Prepare three different batches of ELISA plates according to the established indirect ELISA method. Each ELISA plate is tested with the above-selected Listeria monocytogenes positive and negative quality control products and duplicate wells are set. After the test is completed, the OD value of each well is read using a microplate reader. 450 The coefficient of variation between batches was calculated according to the formula: coefficient of variation CV = (SD ÷ X) × 100%.

[0093] 1.2.10 Compliance Testing

[0094] The indirect ELISA method established by the present invention and the commercial kit were used to detect 24 mouse sera at the same time, and the OD value of each well was read using a microplate reader. 450 The results of the two methods were compared to evaluate the detection effect.

[0095] 2 Results

[0096] 2.1 Construction of PColdⅡ-10His-inlB recombinant expression vector

[0097] Using inactivated Listeria monocytogenes solution as a template, the target gene was amplified using inlB-F / R primers. PCR detection showed that the length of the amplified fragment was consistent with the predicted fragment size, which was 1893 bp ( Figure 1 The recombinant plasmid PColdⅡ-10His-inlB was detected by bacterial liquid PCR. Figure 2 As shown in the figure, a specific band was found at 2083 bp, and the detection result was consistent with the expected result. The above results indicate that the PColdⅡ-10His-inlB recombinant expression vector was successfully constructed.

[0098] 2.2 Expression, identification and purification of InlB protein

[0099] SDS-PAGE electrophoresis of the InlB protein expression bacteria after induction showed that a protein band of about 69 KDa appeared in the supernatant and precipitate samples ( Figure 3), the size is consistent with the expected band, the target band is darker than other impurities and has a higher concentration. The above results show that InlB protein can be expressed in soluble form and inclusion bodies. PAGE electrophoresis and Western Blot (WB) detection were then performed, and the results are shown in Figure 4 The protein size was consistent with the expected protein. To purify the InlB protein, the supernatant after ultrasonication was purified using Ni NTA beads. SDS-PAGE results showed that the InlB protein was successfully purified.

[0100] 2.3 Results of optimization of indirect ELISA reaction conditions

[0101] To establish the optimal reaction conditions for ELISA, the present invention performed serial dilutions on the coating protein concentration and determined that the optimal coating protein concentration was 4 μg / mL, the serum dilution concentration was 1:400, the optimal blocking solution was 10% skim milk powder, the blocking time was 1 h, the primary antibody incubation time was 1 h, the secondary antibody dilution was 1:2500, the secondary antibody incubation time was 1 h, and the color development time was 8 min, as shown in Table 3.

[0102] Table 3 Optimization results of various reaction conditions of indirect ELISA method

[0103]

[0104]

[0105] 2.4 Determination of the judgment criteria for the indirect ELISA method

[0106] 20 negative samples of Listeria monocytogenes were tested under the optimal reaction conditions, and the OD 450 nm values, and OD values of 20 samples were calculated using Graphpad Prism software. 450 nm analysis, negative serum OD 450 The nm value is between 0.201 and 0.315. The calculated X is 0.2507 and SD is 0.0369. 450 nm≥0.361(X+3SD) is positive. 450 nm<0.361 is negative. Taking into account factors such as result error, under the premise of confirming the reliability of the test results, when the OD of the test sample is 450 A value greater than or equal to 0.37 was considered positive, and a value less than 0.37 was considered negative.

[0107] 2.5 Specificity detection

[0108] The indirect ELISA method established by the present invention was used to detect the positive serum of different mice collected. As shown in Table 4, after detecting OD 450nmOnly the serum of Listeria was positive, while the other positive sera of Brucella, Mycobacterium tuberculosis and ε protein of Clostridium perfringens were all negative. The results showed that the indirect ELISA detection method of the present invention is specific.

[0109] Table 4 Indirect ELISA specificity test results

[0110]

[0111] Note: "+" indicates the result is positive, and "-" indicates the result is negative.

[0112] 2.6 Sensitivity testing

[0113] The positive control sample of InlB protein was serially diluted, and the results showed that at a serum dilution of 1:800 OD 450 The value was 0.405, which was still greater than 0.37 and was positive, indicating that the sensitivity of this method was good (Table 5).

[0114] Table 5 Sensitivity test results

[0115]

[0116] 2.7 Repeatability test

[0117] The results showed that the intra-batch coefficient of variation of the ELISA method of the present invention was 2.79% to 6.64%, and the inter-batch coefficient of variation was 2.61% to 7.75% (CV = (standard deviation / mean) × 100%), indicating that the method had good repeatability.

[0118] 2.8 Compliance Testing

[0119] The optimized ELISA method and commercial kit were used to test 24 mouse serum samples. The results showed that the negative coincidence rate was 83.33% (5 / 6), the positive coincidence rate was 100% (18 / 18), and the overall coincidence rate was 95.83% (23 / 24).

[0120] Example 2 Assembly of an indirect ELISA kit for Listeria monocytogenes

[0121] An indirect ELISA kit for detecting Listeria monocytogenes, comprising the following components:

[0122] ELISA plate: The ELISA plate coated with InlB protein prepared in Example 1, with a coating antigen concentration of 4 μg / mL and a blocking solution of 10% skim milk powder;

[0123] Enzyme-labeled secondary antibody: e.g. GoatAnti-Mouse IgG (H+L), HRP Conjugate;

[0124] Diluent: PBST buffer;

[0125] Washing solution: PBS buffer containing 0.1% Tween 20;

[0126] Color development solution: TMB solution;

[0127] Stop solution: 2 mol / L H2SO4 solution;

[0128] Negative quality control: negative quality control serum containing antibodies that are not infected with Listeria monocytogenes;

[0129] Positive quality control product: Listeria monocytogenes InlB protein antibody positive quality control serum.

[0130] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An indirect ELISA kit for detecting antibodies to Listeria monocytogenes, characterized in that: including an ELISA plate coated with an antigen; The antigen is a recombinant InlB protein, and its amino acid sequence is shown in SEQ ID NO.

2.

2. The indirect ELISA kit according to claim 1, wherein The preparation method of the recombinant InlB protein comprises the following steps: The inlB gene was homologously recombined with the PColdⅡ-10His plasmid to obtain the recombinant plasmid PColdⅡ-10His-inlB; The recombinant plasmid PColdⅡ-10His-inlB was transformed into competent Escherichia coli cells to obtain a recombinant expression strain; After inducing expression culture of the recombinant expression strain, the recombinant InlB protein is obtained by protein purification; The nucleotide sequence of the inlB gene is shown in SEQ ID NO.

1.

3. The indirect ELISA kit according to claim 1, wherein The method for preparing the antigen-coated ELISA plate comprises the following steps: The antigen is diluted with a coating solution to obtain a suspension, and the suspension is added to an enzyme-labeled well for coating treatment. A blocking solution is then added for blocking treatment to obtain the antigen-coated enzyme-labeled plate.

4. The indirect ELISA kit according to claim 3, wherein The concentration of the recombinant InlB protein in the suspension was 4 μg / mL.

5. The indirect ELISA kit according to claim 3, wherein The blocking solution is 10% skim milk powder.

6. The indirect ELISA kit according to claim 1, wherein The indirect ELISA kit further comprises an enzyme-labeled secondary antibody, a diluent, a washing solution, a color developing solution, a stop solution, a negative standard substance and a positive standard substance.

7. The indirect ELISA kit according to claim 6, wherein The diluent is PBST buffer; and / or The washing solution is PBS buffer containing 0.1% Tween 20.

8. The indirect ELISA kit according to claim 6, wherein The color developing solution is TMB solution.

9. The indirect ELISA kit according to claim 6, wherein The stop solution is 2 mol / L H2SO4 solution.

10. A method for detecting antibodies to Listeria monocytogenes for non-disease diagnosis purposes, characterized in that: The method comprises the steps of detecting antibodies against Listeria monocytogenes using the indirect ELISA kit according to any one of claims 1 to 9: Add negative control, positive control and sample solution to the antigen-coated ELISA plate respectively for the first incubation reaction; Enzyme-labeled secondary antibody was added to each reaction well for a second incubation reaction; Add color development solution to each reaction well and continue incubation; Add stop solution to each reaction well, measure the absorbance value at a wavelength of 450nm using an enzyme reader, and judge the result.

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