A detection method and kit for detecting listeria monocytogenes

Abstract

The application 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 enzyme label plate coated with an antigen. The amino acid sequence of the antigen is shown as SEQ ID NO. 2. The indirect ELISA detection kit constructed in the application only reacts with positive serum of Listeria monocytogenes, does not cross-react with other serum, and has high specificity. When the serum dilution degree is 1:800, the detection result is still positive, and the sensitivity is high. The batch variation coefficient is between 2.79% and 6.64%, the batch-to-batch variation coefficient is between 2.61% and 7.75%, and the batch-to-batch repeatability is good. For 24 serum samples, the total coincidence rate is 95.83%. The kit provided in the application can effectively detect sample infection of Listeria monocytogenes, and has the advantages of high specificity, high sensitivity and good repeatability.

Description

TECHNICAL FIELD

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

[0002] Listeria monocytogenes, simply referred to as Listeria monocytogenes, is a gram-positive intracellular parasitic pathogenic bacteria that can infect humans and animals. Listeria monocytogenes widely exists in nature, mainly spreads through fecal-oral route by contaminating food (such as non-sterile dairy products, cooked meat, ready-to-eat fruits and vegetables), and can also infect through damaged skin, mucous membrane or placenta. Humans, cattle, sheep and pigs can all be infected with Listeria monocytogenes. Pregnant women, newborns, the elderly and immunodeficient persons are susceptible to infection, and pregnant women can cause miscarriage, stillbirth or neonatal sepsis and meningitis, and immunocompromised persons are prone to develop sepsis and meningitis.

[0003] The virulence factors of Listeria monocytogenes mainly include hemolytic toxin, actin polymerization protein, C-type phospholipase, internalin, cell wall hydrolase, metalloproteinase, amidase and regulatory factor, etc. After Listeria monocytogenes invades the host, it can cause various responses of host cells and induce the host to produce immune response, thereby producing antibodies to resist the invasion of pathogenic bacteria. Therefore, serum detection is an important basis for the diagnosis of the disease. Among them, internalin can increase the ability of Listeria monocytogenes to invade host cells and enhance the virulence of Listeria monocytogenes, and because of its good immunogenicity, it has become an important target for vaccine development and diagnosis. The present application optimizes the expression conditions to make the InlB protein soluble, ensures its natural conformation, and uses the InlB recombinant protein to establish an indirect ELISA method for detecting Listeria monocytogenes disease, thereby providing a reference basis for the monitoring and diagnosis of Listeria monocytogenes disease. SUMMARY

[0004] The purpose of the present application is to provide a detection method and kit for detecting Listeria monocytogenes to solve the problems existing in the prior art. The kit can effectively detect samples infected with Listeria monocytogenes, and has the advantages of strong specificity, high sensitivity and good repeatability.

[0005] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0006] The present application provides an indirect ELISA kit for detecting Listeria monocytogenes antibody, which comprises an enzyme-labeled plate coated with an antigen.

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

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

[0009] The inlB gene is subjected to homologous recombination with the PCold II-10His plasmid to obtain a recombinant plasmid PCold II-10His-inlB;

[0010] The recombinant plasmid PCold II-10His-inlB is transformed into an E. coli competent cell to obtain a recombinant expression strain;

[0011] After the recombinant expression strain is induced and expressed, the recombinant InlB protein is obtained through protein purification treatment.

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

[0013] Further, the preparation method of the enzyme-labeled plate coated with the antigen comprises the following steps:

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

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

[0016] Further, the blocking solution is 10% skimmed milk powder.

[0017] Further, the indirect ELISA kit further comprises an enzyme-labeled secondary antibody, a diluent, a washing solution, a color developing solution, a termination solution, a negative standard and a positive standard.

[0018] Further, the diluent is a PBST buffer solution; and / or

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

[0020] Further, the color developing solution is a TMB solution.

[0021] Further, the termination solution is a 2 mol / L H2SO4 solution.

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

[0023] The negative quality control, the positive quality control and the sample liquid to be detected are added to the enzyme-labeled plate coated with the antigen respectively, and a first incubation reaction is performed;

[0024] An enzyme-labeled secondary antibody is added to each reaction hole, and a second incubation reaction is performed.

[0025] Add chromogenic solution to each reaction well, and continue to incubate;

[0026] Add stop solution to each reaction well, measure the absorbance value at 450nm wavelength by using an enzyme label instrument, and determine the result.

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

[0028] The present application utilizes PCold II-10His plasmid to express InlB recombinant protein in vitro, detects the soluble expression of the protein by SDS-PAGE and Western Blot, and purifies the InlB recombinant protein by using His Ni NTAbeads. Further, by optimizing the coating amount and blocking solution, an indirect ELISA detection method for Listeria monocytogenes is established, and the specificity, sensitivity and repeatability thereof are detected. The results show that after IPTG is 0.5mM, 16℃, 150rpm overnight induction for 16h, the InlB recombinant protein can be expressed in a soluble manner by WB detection. After optimization, when the InlB recombinant protein is coated at 4μg / mL, 10% skim milk powder is blocked at 37℃ for 1h, the serum to be detected is diluted at 1:400, incubated at 37℃ for 1h, the enzyme-labeled secondary antibody is diluted at 1:2500, incubated at 37℃ for 30min, color development is performed for 8min, and the positive and negative critical value is 0.37, the indirect ELISA detection method for Listeria monocytogenes can be established. The present application also finds that the established indirect ELISA detection kit only reacts with positive serum of Listeria monocytogenes, and has no cross reaction with other serum, and has strong specificity; when the serum dilution degree is 1:800, the detection result is still positive, and the sensitivity is high; the batch variation coefficient is between 2.79% and 6.64%, the batch variation coefficient is between 2.61% and 7.75%, and the batch and batch repeatability is good; for 24 serum samples, the total coincidence rate is 95.83%.

[0029] The indirect ELISA method established by the present application has good reactivity with positive serum of Listeria monocytogenes, and has strong specificity, high sensitivity and good repeatability. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0031] Figure 1 PCR amplification product identification results of the inlB gene; wherein, M: DNA relative molecular weight marker; 1: inlB gene amplification fragment; 2: negative control;

[0032] Figure 2 The PCR identification result of the bacterial liquid of the Pcold II-10His-inlB recombinant plasmid; wherein, M: DNA Marker; 1-5: Pcold II-10His-inlB plasmid; 6: negative control;

[0033] Figure 3 The SDS-PAGE analysis result of the induced expression of the InlB recombinant protein; wherein, M: Protein Marker; 1: supernatant after induction; 2: precipitate after induction; 3: bacterial liquid without induction; 4: supernatant after induction of the Pcold II-10His empty vector; 5: precipitate after induction of the Pcold II-10His empty vector;

[0034] Figure 4 The Western blot analysis result of the InlB recombinant protein after purification; wherein, M: Protein Marker; 1: the InlB recombinant protein after purification. DETAILED DESCRIPTION

[0035] Various illustrative embodiments of the present application are now described in detail. The detailed description should be considered in connection with the accompanying drawings, not limiting the aspects, features and embodiments of the application, but merely illustrating certain aspects, features and embodiments thereof.

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

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in the specification are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in the specification is not intended as an admission that the reference is prior art to the application described and claimed herein.

[0038] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative only, and are not intended to limit the scope of the application.

[0039] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” and the like are open-ended terms that are intended to be synonymous, and are generally used to permit for the inclusion of additional nonspecified elements or integers.

[0040] 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, sera and vectors

[0044] Listeria monocytogenes inactivated bacteria solution, positive quality control, and negative quality control were provided by Beijing Agricultural University; mouse brucellosis positive serum, mouse bovine tuberculosis positive serum, mouse Clostridium perfringens positive serum, and pColdII-10His plasmid were preserved by the Public Health and Biological Safety Prevention and Control Team of Beijing Institute of Animal Science and Veterinary Medicine.

[0045] 1.1.2 Main reagents and instruments

[0046] Goat Anti-Mouse IgG (H+L), HRP Conjugate (Beijing Zhunshi Gold Biotechnology Co., Ltd.); Ni NTA beads (Changzhou Tiandi Renhe Biotechnology Co., Ltd.); PrimeSTAR HS DNA Polymerase (Bodipy Biotechnology (Beijing) Co., Ltd.); 2x Rapid Taq Master Mix, ClonExpress MultiS One Step Cloning Kit (Nanjing Novozyme Biological Technology Co., Ltd.); IPTG, gel preparation kit, page gel staining solution, BSA (Beijing Solabio Co., Ltd.); dialysis bag, primary antibody diluent, His-tag primary antibody, and HRP-labeled secondary antibody (Bi Yun Tian Biotechnology); Blue IV Protein Marker (Beijing Zhunshi Gold Biotechnology Co., Ltd.); StarMarker D2000 Plus (Kangrun Biotechnology-Genestar); skimmed milk powder (BD Company); restriction endonuclease, exposure solution, TMB color developing solution, and 96-well detachable enzyme-labeled plate (ThermoFisher Scientific Company).

[0047] PCR amplification instrument T100 Thermal Cycler (Bio-Rad), NanoDrop One (ThermoFisher Scientific), nucleic acid electrophoresis instrument, vertical electrophoresis instrument, electroblotting instrument (BioRad), exposure instrument (Tecan), spectrophotometer (ThermoFisher Scientific), four-dimensional rotary mixer BE-1100 (Haimen Qishanbi Instrument Manufacturing Co., Ltd.), ultrasonic cell pulverizer SCIENTZ-IID (Ningbo Xinzhi Biotechnology Co., Ltd.), etc. are provided by the Animal Biosafety and Public Health Prevention and Control Team of the Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0048] 1.1.3 Gene and protein sequence

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

[0050] SEQ ID NO. 1:

[0051]

[0052] SEQ ID NO. 2:

[0053] MKEKHNPRRKYCLISGLAIIFSLWIIIGNGAKVQAETITVSTPIKQIFPDDAFAETIKDNLKKKSVTDAVTQNELNSIDQIIANNSDIKSVQGIQYLPNVTKLFLNGNKLTDIKPLTNLKNLGWLFLDENKIKDLSSLKDLKKLKSLSLEHNGISDINGLVHLPQLESLYLGNNKITDITVLSRLTKLDTLSLEDNQISDIVPLAGLTKLQNLYLSKNHISDLRALAGLKNLDVLELFSQECLNKPINHQSNLVVPNTVKNTDGSLVTPEIISDDGDYEKPNVKWHLPEFTNEVSFIFYQPVTIGKAKARFHGRVTQPLKEVYTVSYDVDGTVIKTKVEAGTRITAPKPPTKQGYVFKGWYTEKNGGHEWNFNTDYMSGNDFTLYAVFKAETTEKTVNLTRYVKYIRGNAGIYKLPREDNSLKQGTLASHRCKALTVDREARNGGKLWYRLKNIGWTKAENLSLDRYDKMEYDKGVTAYARVRNASGNSVWTKPYNTAGAKHVNKLSVYQGKNMRILREAKTPITTWYQFSIGGKVIGWVDTRALNTFYKQSMEKPTRLTRYVSANKAGESYYKVPVADNPVKRGTLAKYKNQKLIVDCQATIEGQLWYRIRTSSTFIGWTKAANLRAQK.

[0054] 1.2 Method

[0055] 1.2.1 Primer design

[0056] The primers for amplifying the full-length of inlB gene were designed by SnapGene with reference to the L. monocytogenes EGD gene sequence (HG421741) deposited in NCBI: inlB-F and inlB-R, which were synthesized by Beijing GenScript Biotech Co., Ltd. The specific sequences are shown in Table 1.

[0057] Table 1 Primer sequences for amplifying inlB gene

[0058]

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

[0060] The inlB gene fragment was amplified by using the inactivated Listeria monocytogenes bacterial solution as a template and the primer pair inlB-F / R. The PCR reaction system was 50 μL, including 25 μL PrimeSTAR Max DNA Polymerase, 20 μL sterile enzyme-free water, 2 μL of each primer, and 1 μL of template. The above components were mixed and then centrifuged in cis. The PCR amplification reaction program was pre-denaturation at 98°C for 2 min, denaturation at 98°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 90 s, reaction cycle 35 times, final extension at 72°C for 5 min, and storage at 4°C.

[0061] The pColdII-10His empty vector plasmid was double-digested by Kpn I and Hind III. The total enzyme digestion system was 50 μL, including 5 μL of 10×rCutsmart, 1 μg of pColdII-10His empty vector plasmid, 1 μL of Kpn I and Hind III endonuclease, and ddH2O to a total volume of 50 μL. The above components were mixed and then centrifuged in cis, and placed in a 37°C water bath for enzyme digestion reaction for 2 h. Then the inlB gene amplification product and the enzyme digestion product were analyzed by agarose gel electrophoresis, and the correct size band was cut and subjected to agarose gel DNA recovery.

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

[0063] Then 10 μL of the above connection product was transformed into E. coli competent cells, and single colonies were picked for bacterial liquid culture. The primer pair (M13-F / R, see Table 2) was used for PCR identification, and the positive bacterial liquid sample was sequenced. The plasmid extracted from the correct sequencing bacterial liquid sample was named pColdII-10His-inlB, and the detection concentration was 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 PCold II-10His-inlB was transformed into E. coli BL21 competent cells to obtain a recombinant expression strain; the recombinant expression strain was inoculated into 4 mL of 2xYT liquid medium containing ampicillin (100 mg / mL), and was transferred from fresh bacterial liquid into 20 mL of 2xYT liquid medium containing ampicillin (100 mg / mL) to induce expression, the transfer concentration was 1:100, the OD value was 0.6-0.8, 0.5 mM IPTG was added, the induction conditions were 16 h, 150 rpm, and 16°C. 600

[0068] 1.2.4 Protein immunoblotting identification

[0069] The bacterial liquid was centrifuged at 4400 rpm for 8 min, the supernatant was discarded, and 2 mL of PBS was used for washing once, and the supernatant was discarded after centrifugation at the same speed, 1 mL of PBS was added to the bacterial liquid, and the bacterial liquid was transferred to a 2 mL Ep tube for ultrasonic disruption, centrifuged at 12000 rpm for 20 min at 4°C, and the supernatant and precipitate were collected, respectively, and identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (WB). The identification conditions are as follows: 300 mA, membrane transfer for 1.5 h, 5% skimmed milk powder, room temperature shaking bed blocking for 2 h, using PBST to wash the skimmed milk clean, adding His-tag primary antibody (diluted by 1:5000 of primary antibody diluent), incubating at room temperature for 2 h (or incubating overnight at 4°C), washing the membrane using PBST for 3 times on a shaking bed, each time for 5 min, then discarding the PBST, adding Goat Anti-Mouse IgG (H+L), HRP secondary antibody (diluted by 1:5000 of 5% skimmed milk powder), incubating at room temperature for 1 h, repeating the above membrane washing operation, and finally exposing.

[0070] 1.2.5 Purification of InlB protein

[0071] The induction expression was repeated according to the steps of 1.2.3, the His Ni NTAbeads were treated in advance using Lysis Buffer (10 mM imidazole) and ultrapure water, the supernatant containing the expressed protein was hung on the His Ni NTAbeads, and the beads were incubated on a four-dimensional rotator for 4 h, the flow-through liquid was dropped by gravity column, and the OD 280 value was less than 0.01, and then the protein was eluted using Elushin buffer (250 mM imidazole), and the OD 280 ​The elution was stopped when the value was lower than 0.2. 80 μL of the eluted protein sample was taken for PAGE analysis. The rest of the protein was subjected to the next dialysis treatment. Imidazole was replaced with PBS, and the PBS was changed regularly. After 4 times of replacement, the protein was collected. After confirming that the band size was accurate, the band was single, and the purity met the experimental requirements, the next experimental process was entered.

[0072] 1.2.6 Optimization of the conditions of the indirect ELISA method

[0073] Indirect ELISA method:

[0074] (1) The serum sample was diluted with a diluent and mixed thoroughly to obtain a sample solution;

[0075] (2) 100 μL of the diluent, negative quality control, positive quality control, and sample solution were added to the enzyme-labeled holes respectively as blank group, negative control group, positive control group, and experimental group, and duplicate hole experiments were performed, and then incubation was performed at 37°C;

[0076] Negative quality control: negative quality control serum containing antibodies against uninfected Listeria monocytogenes;

[0077] Positive quality control: positive quality control serum of Listeria monocytogenes InlB protein antibodies.

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

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

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

[0081] (6) Add 50 μL of stop solution (2 mol / L H2SO4 solution) to each reaction hole, and then measure the absorbance value of each reaction hole at 450 nm wavelength with an enzyme-labeled instrument after incubation;

[0082] (7) Result determination: the negative and positive quality controls and 30 negative serum samples of Pasteurella multocida were detected, and the OD 450 value was measured by an enzyme-labeled instrument, the average value (X) and the standard deviation (SD) were calculated, the positive critical value (X+3×SD) was calculated, and when the S / P value of the detected sample was greater than X+3×SD, it was determined to be positive, otherwise it was negative.

[0083] The different conditions in the above detection method were optimized:

[0084] Determination of optimal coating concentration of InIB protein. The purified InIB protein was diluted 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, 0.125 μg / mL, 100 μL of the suspension was added to each enzyme-labeled well, and coated overnight at 4°C. The next day, the plate was washed with PBST (2 L PBS phosphate buffer plus 2 mL Tween-20) 5 times, then blocked (5% skim milk, 37°C for 1 hour). The primary antibody (1:400 diluted negative and positive serum from PBST) was added, and incubated at 37°C for 1 hour. After washing the plate, the enzyme-labeled secondary antibody Goat Anti-Mouse IgG (H+L), HRP Conjugate (1:2500 diluted from PBST) was added, and incubated at 37°C for 30 min. After washing the plate, color development was performed (TMB single-component color developing solution) for 10 min in the dark, 50 μL of stop solution was added, and the OD 450 value was detected.

[0085] Determination of optimal blocking solution concentration of InIB protein. The enzyme-labeled plate was coated with InIB protein at a concentration of 1 μg / mL, and blocked with 5% skim milk, 10% skim milk, and 10% BSA, respectively. The ELISA test was performed under uniform conditions, and the OD 450 value was determined to select the optimal blocking solution concentration.

[0086] 1.2.7 Specificity detection

[0087] According to the indirect ELISA method established in 1.2.6, the positive mouse serum of Brucella, the positive mouse serum of Mycobacterium tuberculosis, the positive mouse serum of Clostridium perfringens epsilon protein, and the positive mouse serum of Listeria monocytogenes were detected and repeated wells were set to observe whether InIB protein had cross reaction with other pathogens, so as to judge its specificity.

[0088] 1.2.8 Sensitivity detection

[0089] The positive control was diluted at different concentrations, with serum dilutions of 1:800, 1:400, 1:200, 1:100, and 1:50. The indirect ELISA method established in 1.2.6 was used for detection, and negative controls were set up, with 3 repeats for each gradient. The sensitivity of the method was judged by comparing the P / N value (average OD 450 value of positive control / average OD 450 value of negative control).

[0090] 1.2.9 Reproducibility detection

[0091] Intra-batch repeatability testing: A batch of ELISA plates were coated according to the indirect ELISA method established in 1.2.6. The plates were blocked and washed the following day. Then, 90 μL of 50% glycerol was added to each well, and the plates were incubated at room temperature for 10 min. The glycerol was then removed, and the plates were vacuum-sealed at 4°C. Listeria monocytogenes positive and negative controls were selected, and on days 1, 3, and 5, a single plate was selected for testing the serum samples, with duplicate wells provided. After testing, the OD values ​​of each well were read using a microplate reader. 450 The value is calculated using the formula: Coefficient of Variation (CV) = (SD ÷ X) × 100%.

[0092] Inter-batch repeatability testing: Three different batches of ELISA plates were prepared according to the established indirect ELISA method. Each plate was used to test the selected Listeria monocytogenes positive and negative controls, and duplicate wells were included. After the test, the OD values ​​of each well were read using an ELISA reader. 450 The value is calculated using the formula: Coefficient of Variation (CV) = (SD ÷ X) × 100%. The inter-batch coefficient of variation is then calculated.

[0093] 1.2.10 Conformity Testing

[0094] The indirect ELISA method established in this invention and a commercially available kit were used to simultaneously detect 24 mouse serum samples. The OD values ​​of each well were read using a microplate reader. 450 The results of the two methods are compared to evaluate the detection effectiveness.

[0095] 2 Results

[0096] 2.1 Construction of the 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 amplified fragment length was consistent with the predicted fragment size, at 1893 bp. Figure 1 The recombinant plasmid PColdⅡ-10His-inlB was detected by bacterial culture PCR, as shown... Figure 2 As shown, a specific band was found at 2083bp, consistent with the expected result. These 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 InlB protein-expressing bacteria after induction showed a protein band of approximately 69 kDa in both the supernatant and precipitate samples. Figure 3), the size was consistent with the expected band, and the target band was darker and more concentrated than other impurities. The above results showed that the InlB protein could be expressed in soluble form and in inclusion bodies. Subsequent PAGE electrophoresis and Western Blot (WB) detection showed that the protein was expressed successfully Figure 4 , which was consistent with the expected protein size. To purify the InlB protein, the supernatant after ultrasonic disruption was selected for purification by Ni NTA beads, and the SDS-PAGE results showed that the InlB protein was successfully purified.

[0100] 2.3 Optimization results of indirect ELISA reaction conditions

[0101] To establish the optimal reaction conditions for ELISA, the present application performed a dilution of the coating protein concentration by a factor of two, and determined that the optimal coating protein concentration was 4 μg / mL, the serum dilution concentration was 1:400, the optimal blocking liquid was 10% skimmed 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 each reaction condition of the indirect ELISA method

[0103]

[0104]

[0105] 2.4 Determination of the determination criteria of the indirect ELISA method

[0106] Under the optimal reaction conditions, 20 samples of negative Listeria monocytogenes were detected, and the OD 450 nm value was obtained. The OD 450 nm of the 20 samples was analyzed using Graphpad Prism software, and the OD 450 nm value of the negative serum was 0.201-0.315. Through calculation, X was 0.2507 and SD was 0.0369. When the OD 450 nm of the detected sample was ≥0.361 (X+3SD), it was positive, and when the OD 450 nm of the detected sample was <0.361, it was negative. Considering the result error and other factors, under the premise of confirming the reliability of the detection results, when the OD 450 value of the detected sample was greater than or equal to 0.37, it was judged to be positive, and when it was less than 0.37, it was judged to be negative.

[0107] 2.5 Specific detection

[0108] The indirect ELISA method established by the present application was used to detect different positive mouse sera collected. As shown in Table 4, the OD 450nmOnly the serum of Listeria monocytogenes was positive, and the other sera of Brucella, Mycobacterium tuberculosis and epsilon protein of Clostridium perfringens were negative, which showed that the indirect ELISA detection method of the application had specificity.

[0109] Table 4: Results of specificity test of indirect ELISA

[0110]

[0111] Note: "+" represents that the result is determined as positive, and "-" represents that the result is determined as negative.

[0112] 2.6 Sensitivity test

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

[0114] Table 5: Results of sensitivity test

[0115]

[0116] 2.7 Reproducibility test

[0117] The results showed that the intra-batch reproducibility coefficient of the ELISA method of the application was 2.79% to 6.64%, the inter-batch reproducibility coefficient was 2.61% to 7.75% (CV = (standard deviation / average value) x 100%), which indicated that the reproducibility of the method was good.

[0118] 2.8 Compliance test

[0119] The optimized ELISA detection method and the commercial kit were used to detect 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 indirect ELISA kit for Listeria monocytogenes disease

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

[0122] An enzyme-labeled plate: the enzyme-labeled plate coated with InlB protein prepared in Example 1, the concentration of the coated antigen was 4 μg / mL, and the blocking solution was 10% skimmed milk powder;

[0123] An enzyme-labeled secondary antibody: for example, Goat Anti-Mouse IgG (H+L), HRP Conjugate;​

[0124] Dilution solution: PBST buffer solution;

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

[0126] Color developing solution: TMB solution;

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

[0128] Negative quality control: antibody-negative quality control serum containing uninfected Listeria monocytogenes;

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

[0130] The above-described embodiments are merely preferred modes of the present application and are not intended to limit the scope of the present application, and various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the protection scope of the present application as defined by the claims.

Claims

1. An indirect ELISA kit for detecting antibodies to Listeria monocytogenes, characterized in that, The enzyme-labeled plate coated with the antigen; The antigen is a recombinant InlB protein, and the amino acid sequence is shown as SEQ ID NO. 2; The indirect ELISA kit further comprises an enzyme-labeled secondary antibody, a diluent, a washing solution, a color developing solution, a termination solution, negative quality control samples, and positive quality control samples.

2. The indirect ELISA kit according to claim 1, characterized in that, The preparation method of the recombinant InlB protein comprises the following steps: The inlB The gene was homologously recombined with the PCold II-10His plasmid to obtain a recombinant plasmid PCold II-10His-inlB. The recombinant plasmid PColdII-10His-inlB is transformed into the competent cells of Escherichia coli to obtain a recombinant expression strain; After the recombinant expression strain is induced and expressed, the recombinant InlB protein is obtained through protein purification treatment. The inlB The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

3. The indirect ELISA kit according to claim 1, characterized in that, The preparation method of the enzyme-labeled plate coated with the antigen comprises the following steps: The antigen is diluted with a coating solution to obtain a suspension, the suspension is added to the enzyme-labeled holes for coating treatment, then a blocking solution is added for blocking treatment, and the enzyme-labeled plate coated with the antigen is obtained.

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

5. The indirect ELISA kit according to claim 3, characterized in that, The blocking solution is 10% skimmed milk powder.

6. The indirect ELISA kit according to claim 1, characterized in that, The diluent is a PBST buffer solution; and / or The washing solution is a PBS buffer solution containing 0.1% Tween 20.

7. The indirect ELISA kit according to claim 1, characterized in that, The color developing solution is a TMB solution.

8. The indirect ELISA kit according to claim 1, characterized in that, The termination solution is a 2 mol / L H2SO4 solution.

9. A method for detecting antibodies to Listeria monocytogenes for non-disease diagnostic purposes, characterized in that, The method comprises the steps of detecting the antibody of Listeria monocytogenes by using the indirect ELISA kit according to any one of claims 1-8. The negative quality control samples, the positive quality control samples, and the sample liquid to be detected are added to the enzyme-labeled plate coated with the antigen for the first incubation reaction; The enzyme-labeled secondary antibody is added to each reaction hole for the second incubation reaction; The color developing solution is added to each reaction hole for continuous incubation; The termination solution is added to each reaction hole, the absorbance value is measured at 450 nm wavelength by using an enzyme-labeled instrument, and the result is determined.

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