Sample diluent, application of sample diluent in bovine paratuberculosis screening kit and product

Abstract

The invention discloses a sample diluent, application of the sample diluent in a bovine paratuberculosis screening kit and a product, and relates to the technical field of biology. The sample diluent is prepared from 20 to 30 mu g / mL of mycobacterium phlei extract, 40 to 60 mM of PBS (Phosphate Buffer Solution) and 0.8 to 1.0 percent of NaCl. The invention further provides a chemiluminescence detection kit for screening bovine paratuberculosis, the chemiluminescence detection kit comprises the sample diluent, and the chemiluminescence detection kit has the characteristics of high throughput, high speed, high specificity and accurate result, is suitable for large-scale screening, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a sample diluent and its application in a bovine paratuberculosis screening kit and a product. Background Art

[0002] Bovine paratuberculosis, also known as "paratuberculous enteritis", refers to a chronic infectious disease of the digestive tract caused by infection with Mycobacterium paratuberculosis. The bacterium is usually a short rod with a length of 0.5μm-1.5μm. It is a Gram-positive bacterium with filamentous branches, no flagella, no capsule, no spores and no motility. It has strong resistance to the external environment and can survive for several months. The late stage of infection is mainly characterized by intermittent diarrhea and intractable diarrhea, and gradually becomes emaciated and depressed. The milk secretion of lactating cows gradually decreases or even stops, and they gradually die of exhaustion.

[0003] The incubation period of bovine paratuberculosis is long, and the early symptoms are not obvious. The disease can infect calves and breeding cattle, and once infected, it will cause huge economic losses. Therefore, regular screening and quarantine of farms are particularly important. At present, the methods of laboratory diagnosis include microscopy, allergy, enzyme-linked immunosorbent assay, and PCR. Mycobacterium paratuberculosis has many common components with Mycobacterium phlei, Mycobacterium kansasii, and Mycobacterium fortuitum. Cross-reactions often occur during serological diagnosis. In addition, some of the above methods have problems such as cumbersome processes, long time consumption, and high costs. They are not suitable for large-scale screening. There is a lack of high-throughput, fast, high-specificity, and accurate detection systems on the market for screening reagents for Mycobacterium paratuberculosis infection.

[0004] Chinese patent CN105907770A discloses a recombinant gene, a recombinant protein encoded by the recombinant gene, its application, and a detection kit and method for bovine paratuberculosis. The kit for detecting bovine paratuberculosis serum described in the invention includes a recombinant protein, a coating solution, a washing solution, a blocking solution, a diluent, a rabbit anti-bovine IgG and a substrate colorimetric solution. The invention uses the recombinant protein obtained by encoding the recombinant gene as a coating antigen and adopts indirect ELISA to detect bovine paratuberculosis. The detection specificity is 91%, the sensitivity is 87%, and the compliance rate is 89%, and the detection compliance rate is low.

[0005] There are few studies on the screening of bovine paratuberculosis in the prior art. The public indirect ELISA method is time-consuming and has a low degree of automation, which is not conducive to sample screening. There is an urgent need to provide a screening reagent for Mycobacterium paratuberculosis infection that has a simple detection method, fast speed, and high accuracy of detection results. Summary of the invention

[0006] The invention aims to provide a sample diluent and its application in a bovine paratuberculosis screening kit and a product.

[0007] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows: In one aspect, the present invention provides a sample diluent, comprising 20-30 ug / mL Mycobacterium phlei extract, 40-60 mM PBS and 0.8-1.0% NaCl.

[0008] Furthermore, the sample diluent includes 20 ug / mL Mycobacterium phlei extract, 50 mM PBS and 0.9% NaCl.

[0009] Specifically, the concentration of NaCl is a mass volume ratio concentration.

[0010] Specifically, the preparation of the Mycobacterium phlei extract comprises the following steps: (1) inoculating Mycobacterium phlei into a fermentation medium for fermentation to obtain a fermentation liquid; (2) The obtained fermentation broth is inactivated with formaldehyde, centrifuged, the bacterial cells are collected, and the treatment liquid is added. After treatment at 80-90° C. for 0.4-1 h, ultrasonication, centrifugation, and filtration are performed to obtain the Mycobacterium phlei extract.

[0011] Furthermore, before inoculating Mycobacterium phlei into the fermentation medium, the method further includes the step of inoculating Mycobacterium phlei into slant culture and seed culture.

[0012] According to some embodiments of the present invention, the slant culture medium includes: 0.3 g soy peptone, 0.3 g tryptone, 0.3 g yeast extract, 0.4 g glucose, 0.4 g sucrose, 0.06 g magnesium sulfate, 0.06 g disodium hydrogen phosphate, 0.06 g potassium dihydrogen phosphate, 0.04 g sodium chloride, 1.8 g agar, add distilled water to 100 mL, pH 7.5, and sterilize at 121°C for 30 min.

[0013] Furthermore, the slant culture is to inoculate Mycobacterium phlei into the slant culture medium and place it at 35-37° C. for 72-96 hours to activate the strain.

[0014] Furthermore, the temperature of the slant culture is 35°C.

[0015] According to some embodiments of the present invention, the seed culture medium includes: 0.4 g soy peptone, 0.4 g tryptone, 0.4 g yeast extract, 0.4 g glucose, 0.4 g sucrose, 0.04 g magnesium sulfate, 0.06 g disodium hydrogen phosphate, 0.06 g potassium dihydrogen phosphate, 0.04 g sodium chloride, 0.6 g glycerol, add distilled water to 100 mL, pH 7.5, and sterilize at 121°C for 30 min.

[0016] Furthermore, the seed culture is to inoculate Mycobacterium phlei into a seed culture medium, and culture it at 35-37° C. and a rotation speed of 180-220 rpm for 48-96 hours to obtain a seed solution.

[0017] Furthermore, the seed culture conditions are 35° C. and 200 rpm.

[0018] Furthermore, the fermentation medium described in step (1) comprises 2-6 g / L soy peptone, 2-6 g / L tryptone, 2-6 g / L yeast extract, 2-6 g / L glucose, 2-6 g / L sucrose, 0.4-0.6 g / L magnesium sulfate, 0.4-0.8 g / L disodium hydrogen phosphate, 0.4-0.8 g / L potassium dihydrogen phosphate, 0.4-0.8 g / L sodium chloride, and 8-12 g / L glycerol.

[0019] Furthermore, the fermentation medium in step (1) comprises 4 g / L soy peptone, 4 g / L tryptone, 4 g / L yeast extract, 4 g / L glucose, 4 g / L sucrose, 0.5 g / L magnesium sulfate, 0.6 g / L disodium hydrogen phosphate, 0.6 g / L potassium dihydrogen phosphate, 0.6 g / L sodium chloride, and 10 g / L glycerol.

[0020] Specifically, the seed solution prepared above is inoculated into a fermentation medium for culturing; Furthermore, the inoculation amount is 5-10% (v / v).

[0021] Furthermore, the fermentation culture in step (1) is carried out under the conditions of 35-38°C, a rotation speed of 120-180 rpm, and a ventilation ratio of 1:(0.8-2) for 48-96 hours; Furthermore, the fermentation culture conditions in step (1) are aeration culture at 35°C, a rotation speed of 150 rpm, and a ventilation ratio of 1:1.

[0022] Furthermore, the concentration of formaldehyde in step (2) is 0.2-0.6%; furthermore, the concentration of formaldehyde is 0.4%.

[0023] Further, in step (2), the volume ratio of the fermentation liquid to formaldehyde is 1:(0.8-2); Furthermore, in step (2), the volume ratio of the fermentation liquid to formaldehyde is 1:1.

[0024] Furthermore, the mass volume ratio of the wet weight of the bacterial cells to the treatment liquid in step (2) is 1:(3-5); Furthermore, the mass volume ratio of the wet weight of the bacteria to the treatment liquid described in step (2) is 1:4.

[0025] Further, the treatment solution has a formula of 10-20 mM PBS, 0.5-2 mM EDTA, 40-60 mM sodium glutamate, a final concentration of 0.2-0.4% glycine, 1-2% sucrose, 1-3% trehalose, 1-2% mannitol, 0.05-0.1% Tween-20, 0.2-0.4% poloxamer, 0.4-0.6% CHAPS and 0.1-0.2% SDS, pH 6-8; Furthermore, the treatment solution has a formula of 20 mM PBS, 1 mM EDTA, 50 mM sodium glutamate, a final concentration of 0.3% glycine, 1% sucrose, 2% trehalose, 1.5% mannitol, 0.1% Tween-20, 0.3% poloxamer, 0.5% CHAPS and 0.1% SDS, pH 7.2.

[0026] Furthermore, in step (2), the treatment liquid is added and then treated at 85° C. for 0.5 h.

[0027] Furthermore, in step (2), ultrasonication is performed after the heating treatment, with an ultrasonic power of 100-200 W, ultrasonication for 1-3 s, pause for 2-4 s, a total duration of 15-20 min, and an ultrasonic temperature of 2-8°C.

[0028] Furthermore, after ultrasonic disruption in step (2), the mixture is centrifuged at 5000-8000 g for 15-25 min to remove the precipitate, and the supernatant is collected and filtered through a 0.2 μm filter membrane.

[0029] In yet another aspect, the present invention provides use of the above-mentioned sample diluent in preparing a bovine paratuberculosis screening kit.

[0030] In yet another aspect, the present invention provides a chemiluminescent detection kit for bovine paratuberculosis screening, wherein the chemiluminescent detection kit comprises the above-mentioned sample diluent.

[0031] Specifically, the chemiluminescent detection kit further comprises a biotin-labeled Mycobacterium paratuberculosis antigen, an acridine sulfonamide-labeled mouse anti-bovine IgG antibody, and streptavidin magnetic beads.

[0032] Furthermore, the chemiluminescence detection kit also includes standard products and quality control products.

[0033] Furthermore, the working concentration of the biotin-labeled Mycobacterium paratuberculosis antigen is 0.2-1 ug / mL; Furthermore, the working concentration of the biotin-labeled Mycobacterium paratuberculosis antigen is 0.5 ug / mL.

[0034] Furthermore, the method for preparing the biotin-labeled Mycobacterium paratuberculosis antigen comprises the following steps: (1) diluting the Mycobacterium paratuberculosis antigen with a buffer solution; (2) Add biotin to the antigen dilution in (1); (3) centrifuging and ultrafiltering the labeled product obtained in step (2), and collecting the ultrafiltration product; (4) Add HRP conjugate stabilizer / diluent IV and filter to obtain biotin-labeled Mycobacterium paratuberculosis antigen.

[0035] Furthermore, the buffer solution in step (1) is 0.02 M PB buffer solution, and the pH of the buffer solution is 7.2.

[0036] Furthermore, the biotin in step (2) is Sulfo-NHS-LC-Biotin.

[0037] Furthermore, the centrifugation conditions in step (3) are 2-8°C, the centrifugal force is 4900-5000g, and the centrifugation time is 10-15min.

[0038] Furthermore, the working concentration of the acridine sulfonamide-labeled mouse anti-bovine IgG antibody is 0.5-1.2 ug / mL; Furthermore, the working concentration of the acridine sulfonamide-labeled mouse anti-bovine IgG antibody is 1 ug / mL.

[0039] Furthermore, the method for preparing the acridine sulfonamide-labeled mouse anti-bovine IgG antibody comprises the following steps: (1) Dilute the mouse anti-bovine IgG antibody with buffer; (2) adding acridine sulfonamide to the antigen dilution in (1); (3) centrifuging and ultrafiltering the labeled product obtained in step (2), and collecting the ultrafiltration product; (4) Add HRP conjugate stabilizer / diluent IV and filter to obtain acridine sulfonamide-labeled mouse anti-bovine IgG antibody.

[0040] Furthermore, the buffer solution in step (1) is 0.05 M CBS buffer solution.

[0041] Furthermore, the centrifugation conditions in step (3) are 2-8°C, the centrifugal force is 4900-5000g, and the centrifugation time is 10-15min.

[0042] Furthermore, the concentration of the streptavidin magnetic beads is 0.2-1.0 mg / mL; Furthermore, the concentration of the streptavidin magnetic beads is 0.4 mg / mL.

[0043] Furthermore, the preparation method of the streptavidin magnetic particles is: take streptavidin-magnetic particles and use a magnetic separator to perform magnetic adsorption, add HRP conjugate stabilizer / diluent IV and mix the magnetic beads to obtain.

[0044] Specifically, the biotin-labeled Mycobacterium paratuberculosis antigen is a genetically recombinant antigen, and the length of the antigen amino acid sequence is 266 aa.

[0045] Furthermore, the antigen sequence is shown in SEQ ID NO:1.

[0046] SEQ ID NO: 1: MNALARLRGRKVVPQRSDGELDAMAAAGAVVAAALQAVRAAAVAGVSTLSLDQIAESVIRDAGAVPSFLGYHGYPASICASVNDRVVHGIPSAAEVLAPGDLVSIDCGAVLDGWHGDAAITFGVGALDP ADEALCQGTRESLEAGIAAMVPGNRLTDVSHAIELGTRAAETRHGRAFGIVEGYGGHGIGRQMHMDPFLPNEGSPGRGPLLAPGSVLAIEPMLTLGTGKTVVLDDQWTVITTDGSRAAHWEHTVAVTDAG PRILTIG.

[0047] Furthermore, the standard products include low-value standard products and high-value standard products.

[0048] Further, the low-value standard is a solution containing 60AU / mL Mycobacterium paratuberculosis antibodies; Furthermore, the high-value standard is a solution containing 480AU / mL Mycobacterium paratuberculosis antibodies.

[0049] Specifically, the standard substance is used to calibrate the master curve of the reagent.

[0050] Furthermore, the quality control products include negative quality control products and positive quality control products.

[0051] Further, the negative control product is a buffer solution that does not contain Mycobacterium paratuberculosis antibodies; Furthermore, the positive quality control product is a solution containing 240AU / mL Mycobacterium paratuberculosis antibodies.

[0052] Specifically, the quality control product is used to evaluate whether the kit is under control.

[0053] In yet another aspect, the present invention provides use of a chemiluminescent detection kit in screening for bovine paratuberculosis.

[0054] Specifically, the determination result of the chemiluminescent detection kit of the present invention is: if the detection result is ≥120 AU / mL, it is positive; if the detection result is <90 AU / mL, it is negative; if the detection result is between 90-120 AU / mL, it is a gray area.

[0055] The beneficial effects of the present invention are: The sample diluent of the present invention can reduce the interference of other IgG antibodies. The chemiluminescence detection kit for bovine paratuberculosis screening provided by the present invention comprises the above-mentioned sample diluent, biotin-labeled Mycobacterium paratuberculosis antigen, streptavidin magnetic particles, and acridine sulfonamide-labeled secondary antibody; the signal amplification system of biotin and streptavidin improves the sensitivity of the kit. The detection kit of the present invention has the characteristics of high throughput, fast speed, high specificity, and accurate results, and is suitable for large-scale screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is the electrophoresis diagram of Mycobacterium paratuberculosis antigen. In the figure, M1 is a marker, BSA is bovine serum albumin, and R is a Mycobacterium paratuberculosis antigen. DETAILED DESCRIPTION

[0057] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0058] Example 1 Preparation of Mycobacterium paratuberculosis antigen Preparation steps: (1) Search for the specific amino acid sequence fragment of Mycobacterium paratuberculosis from GeneBank. The sequence is as follows: MNALARLRGRKVVPQRSDGELDAMAAAGAVVAAALQAVRAAAVAGVSTLSLDQIAESVIRDAGAVPSFLGYHGYPASICASVNDRVVHGIPSAAEVLAPGDLVSIDCGAVLDGWHGDAAITFGVGALDP ADEALCQGTRESLEAGIAAMVPGNRLTDVSHAIELGTRAAETRHGRAFGIVEGYGGHGIGRQMHMDPFLPNEGSPGRGPLLAPGSVLAIEPMLTLGTGKTVVLDDQWTVITTDGSRAAHWEHTVAVTDAG PRILTIG.

[0059] (2) The amino acid sequence is codon optimized according to the species preference during protein expression. The optimized DNA sequence is shown in SEQ ID NO: 2.

[0060] SEQ ID NO:2: .

[0061] (3) Design primers for gene amplification and perform gene amplification.

[0062] (4) The gene was double-digested, the digestion product was purified, and the purified gene fragment was ligated with the vector pET28a(+) using T4 ligase, transformed into competent Escherichia coli DH5α, spread on a plate containing ampicillin, and cultured at 37°C overnight.

[0063] (5) Screen the positive clone recombinant plasmid and transform it into competent E. coli BL21, add IPTG to induce expression. The induced protein is purified by Ni-NTA. The protein gel run is shown in the figure below: Figure 1 .

[0064] (6) The protein content of the purified protein was measured using an ultraviolet spectrophotometer, and the concentration was 2.15 mg / mL.

[0065] Example 2 Preparation of biotin-labeled antigen (1) Place 1 mg of bovine Mycobacterium paratuberculosis antigen in a pyrogen-free vial and dilute the antigen to 1 mg / mL using 0.02 M PB (pH 7.2) buffer.

[0066] (2) Add 5.5 μL of 10 mg / mL Sulfo-NHS-LC-Biotin to 1 mL of the above antibody dilution, mix well, and place at room temperature for 2 hours away from light.

[0067] (3) Add the labeled product to a 10 kD ultrafiltration tube, and then use 0.02 M PB (pH 7.2) buffer to make up the total volume of the liquid to 10 mL. Place it in a high-speed centrifuge for balance. Set the centrifuge temperature to 2-8°C, the centrifugal force to 4900-5000g, and the centrifugation time to 10-15 min. After centrifugation, observe the residual volume of the recovered liquid. If the residual volume is greater than 1 mL, continue centrifugation until the residual volume is less than or equal to 1 mL. If the residual volume is less than or equal to 1 mL, continue ultrafiltration 3 times according to the above steps.

[0068] (4) Collect the ultrafiltration product and record the volume, convert the biotin-labeled Mycobacterium paratuberculosis antigen concentration, and adjust the concentration of biotin-labeled Mycobacterium paratuberculosis antigen to 0.5 ug / mL using commercial HRP conjugate stabilizer / diluent IV. Use a diaphragm vacuum pump to filter the prepared biotin-labeled Mycobacterium paratuberculosis antigen solution through a 0.22 μm filter membrane and set aside.

[0069] Example 3 Preparation of Acridine Sulfonamide Labeled Antibody (1) Place 1 mg of mouse anti-bovine IgG antibody in a pyrogen-free vial and dilute the mouse anti-bovine IgG to 1 mg / mL with 0.05 M CBS buffer; (2) Take 20 μL of 2 mg / mL acridine sulfonamide (NSP-SA-NHS) solution and add it to the above antibody dilution solution, mix well, and place at room temperature for 2 hours away from light.

[0070] (3) Add the labeled product to a 30 kD ultrafiltration tube, and then use 0.02 M PB (pH 7.2) buffer to make up the total volume of the liquid to 10 mL. Place it in a high-speed centrifuge for balance. Set the centrifuge temperature to 2-8°C, the centrifugal force to 4900-5000g, and the centrifugation time to 10-15 min. After centrifugation, observe the residual volume of the recovered liquid. If the residual volume is greater than 1 mL, continue centrifugation until the residual volume is less than or equal to 1 mL. If the residual volume is less than or equal to 1 mL, continue ultrafiltration 3 times according to the above steps.

[0071] (4) Collect the ultrafiltration product and record the volume, convert the concentration of acridine sulfonamide-labeled antibody, and adjust the concentration of acridine sulfonamide-labeled antibody to 1 ug / mL using commercial HRP conjugate stabilizer / diluent IV (purchased from Huzhou Yingchuang Biotechnology Co., Ltd.). Filter the prepared acridine sulfonamide-labeled antibody solution using a 0.22 μm filter membrane using a diaphragm vacuum pump and set aside.

[0072] Example 4 Preparation of Streptavidin Magnetic Particles Place 40 mL of 10 mg / mL streptavidin-magnetic particles in a 50 mL centrifuge tube, place the centrifuge tube on a suitable 50 mL magnetic separator, magnetically adsorb until the solution is clear, let stand for 2 min, discard the clear solution, remove the 50 mL centrifuge tube, add 40 mL HRP conjugate stabilizer / diluent IV and mix the magnetic beads. Repeat this washing operation with HRP conjugate stabilizer / diluent IV for a total of three times, and keep the last clear solution. Then add it to 960 mL HRP conjugate stabilizer / diluent IV, with a final concentration of 0.40 mg / mL, and set aside.

[0073] Example 5 Preparation of Standards and Quality Controls (1) Positive serum: purchased from Prodao (Beijing) Standard Technology Co., Ltd.; (2) Dilute the serum with the calibration diluent in multiples of 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, and 1:640. After dilution, test with the IDEXX Mycobacterium paratuberculosis Avm subspecies antibody detection kit.

[0074] The results are as follows: Table 1

[0075] (3) Built-in main curve setting Assemble qualified biotin-labeled antibodies, acridine sulfonamide-labeled antibodies and magnetic particles into reagents and place them into the applicable machine DNK-C500; Place 8 working calibrants of 0, 15, 30, 60, 120, 240, 480, and 960 AU / mL into the sample rack for testing on the machine. Each point needs to be repeated 3 times. Outliers were removed, and the average light value of the eight working calibrants (Y) was fitted with the labeled concentration (X) using a four-parameter double logarithm (logX-logY) as the built-in master curve.

[0076] The above-mentioned gradient calibrants diluted in multiple ratios were tested with the reagents prepared in Examples 1 to 4, and the automatic detection process of the luminometer was as follows: the instrument aspirated 50 μL of the calibrants, added 40 μL of magnetic particles and 50 μL of biotin-labeled bovine paratuberculosis antigen, mixed, reacted at 37° C. for 8 minutes, washed, and then added 100 μL of acridine sulfonamide-labeled anti-bovine IgG antibody, mixed, reacted at 37° C. for 8 minutes, washed, and finally added the luminescent substrate, and the instrument automatically read the luminescence value and calculated the concentration.

[0077] The results are as follows: Table 2

[0078] (4) Preparation of product standards Preparation of low-value calibrator (60AU / mL): Dilute serum at a ratio of 1:160 with 50mM PBS buffer containing 0.2% Tween 20 and 0.5% bovine serum albumin, mix well, mark and record accordingly.

[0079] Preparation of high-value calibrator (480AU / mL): Dilute serum at a ratio of 1:20 with 50mM PBS buffer containing 0.2% Tween 20 and 0.5% bovine serum albumin, mix well, mark and record accordingly.

[0080] (5) Product calibration material assignment The reagents prepared in the above Examples 1-3 were installed, and the low-value calibrators and high-value calibrators were assigned values ​​on the fully automatic chemiluminescence immunoassay analyzer Venus 100S of Clase Biotech (Chongqing) Co., Ltd. Two devices were used, each device was tested for 3 days, 10 data were tested every day, and 60 data were tested for each calibrator. After the outliers were proposed, the average concentration was calculated as the assigned concentration. The assigned concentration of the low-value calibrator in this example was 60.5 AU / mL, and the assigned concentration of the high-value calibrator was 481 AU / mL.

[0081] (6) Preparation and assignment of product quality control materials Preparation of negative control: Use 50mM PBS buffer containing 0.2% Tween 20 and 0.5% bovine serum albumin as negative control, and make appropriate marks and records.

[0082] Preparation of positive quality control (240AU / mL): Dilute the serum at a ratio of 1:40 with 50mM PBS buffer containing 0.2% Tween 20 and 0.5% bovine serum albumin, mix well, mark and record accordingly.

[0083] The reagents prepared in the above-mentioned Examples 1 to 3 were installed, and the low-value calibrators and high-value calibrators were assigned on the chemiluminescent immunoassay analyzer. Two devices were used, each device was tested for 3 days, 10 data were tested every day, and 60 data were tested for each quality control product. After the outliers were proposed, the average concentration was calculated as the assigned concentration. The assigned concentration of the negative quality control product in this embodiment is 1.5 AU / mL, and the assigned concentration of the positive quality control product is 239 AU / mL.

[0084] Example 6 Reagent Packaging and Preparation of Kit The main reagents of the test kit are divided, labeled and packaged according to the process.

[0085] The composition of the Mycobacterium paratuberculosis antibody detection kit is as shown in Table 3: Table 3

[0086] Example 7 Preparation of Mycobacterium phlei extract (1) Slant culture Slant culture medium (100 mL): 0.3 g soy peptone, 0.3 g tryptone, 0.3 g yeast extract, 0.4 g glucose, 0.4 g sucrose, 0.06 g magnesium sulfate, 0.06 g disodium hydrogen phosphate, 0.06 g potassium dihydrogen phosphate, 0.04 g sodium chloride, 1.8 g agar, balance distilled water, pH 7.5, moist heat sterilization at 121°C for 30 min.

[0087] After inoculating 20 μL of Mycobacterium phlei (purchased from Shangcheng Beina Chuanglian Biotechnology Co., Ltd., strain number BNCC364386) bacterial solution into the slant culture medium, it was placed in a 35°C biochemical incubator and allowed to stand for 72-96 hours for bacterial activation.

[0088] (2) Seed cultivation Seed culture medium (100 mL): 0.5 g soy peptone, 0.5 g tryptone, 0.4 g yeast extract, 0.4 g glucose, 0.4 g sucrose, 0.04 g magnesium sulfate, 0.06 g disodium hydrogen phosphate, 0.06 g potassium dihydrogen phosphate, 0.04 g sodium chloride, 0.6 g glycerol, balance distilled water, pH 7.5, moist heat sterilization at 121°C for 30 min.

[0089] The activated Mycobacterium phlei was cultured in a seed culture medium at 35° C. and a rotation speed of 200 rpm for more than 48 hours to obtain a seed solution.

[0090] (3) Fermentation culture Fermentation medium (1000 mL): 4 g soy peptone, 4 g tryptone, 4 g yeast extract, 4 g glucose, 4 g sucrose, 0.5 g magnesium sulfate, 0.6 g disodium hydrogen phosphate, 0.6 g potassium dihydrogen phosphate, 0.6 g sodium chloride, 10 g glycerol, balance distilled water, pH 7.5, moist heat sterilization at 121°C for 30 min.

[0091] The seed liquid was inoculated into the fermentation medium at an inoculation rate of 8% (v / v), and cultured with aeration at 35°C, 150 rpm, and 1:1 ratio for 48 h. The fermentation was stopped when the OD600 value did not change much, and the fermentation liquid of Mycobacterium phlei was obtained.

[0092] (4) Preparation of Mycobacterium phlei extract Prepare treatment solution (1000 mL): 7.16 g disodium hydrogen phosphate dodecahydrate, 0.48 g potassium dihydrogen phosphate, 0.29 g EDTA, 8.45 g sodium glutamate, 3 g glycine, 10 g sucrose, 20 g trehalose, 1.5 g mannitol, 1 mL of Tween-20, 3 g poloxamer, 5 g CHAPS, 1 g SDS, balance distilled water, pH 7.2.

[0093] The cultured fermentation liquid was mixed with 0.4% formaldehyde in a volume ratio of 1:1 to inactivate the bacteria for 8-12 h, and the bacteria were collected by centrifugation and washed 3 times with 20 mM PBS. The bacteria were divided into 4 portions, and distilled water, 20 mM PBS, 0.5% SDS and the above treatment solution were added to each portion of the bacteria at a mass volume ratio of wet weight to solution of 1:4. After being treated at 85°C for 0.5 h, the mixture was evenly mixed and ultrasonically disrupted at an ultrasonic power of 150 W for 2 s, paused for 3 s, and a total time of 18 min, ultrasonic temperature is 2-8℃; centrifuge at 6000g for 20min to remove the precipitate, collect the supernatant and filter through 0.2μm filter membrane to obtain the extract of Mycobacterium phlei. The protein content is determined by ultraviolet spectrophotometer. The protein concentration of Mycobacterium phlei extract treated with distilled water is 11.5mg / mL, the protein concentration of Mycobacterium phlei extract treated with 20mM PBS is 12.3mg / mL, the protein concentration of Mycobacterium phlei extract treated with 0.5% SDS is 13.5mg / mL, and the protein concentration of Mycobacterium phlei extract treated with homemade treatment solution is 14.8 mg / mL. After preparation, store at 4℃. If it needs to be stored for a long time, 0.1% PC300 can be added to the Mycobacterium phlei extract.

[0094] Example 8 Validation of the use of Mycobacterium phlei extract for sample dilution 1. Sample collection: (1) IDEXX reagent (purchased from IDEXX Maine Biological Products Trading (Shanghai) Co., Ltd., catalog number P07130-5) was used to screen out 5 samples with negative bovine paratuberculosis antibodies and 5 samples with positive bovine paratuberculosis antibodies; (2) The above negative samples and positive samples were mixed separately to obtain two mixed samples. The mixed negative sample was named NM001, and the mixed positive sample was named PM001.

[0095] 2. Preparation of diluent: The four Mycobacterium phlei extracts prepared from different bacterial cell treatment solutions in Example 7 were diluted with distilled water to a concentration of 20 μg / mL to prepare four sample dilution solutions, and distilled water was used as a comparison.

[0096] Sample diluent 01: Distilled water-treated Mycobacterium phlei extract was diluted with distilled water to a concentration of 20 μg / mL.

[0097] Sample diluent 02: PBS-treated Mycobacterium phlei extract was diluted with distilled water to a concentration of 20 μg / mL.

[0098] Sample diluent 03: SDS-treated Mycobacterium phlei extract was diluted with distilled water to a concentration of 20 μg / mL.

[0099] Sample diluent 04: The Mycobacterium phlei extract treated with the homemade treatment solution was diluted with distilled water to a concentration of 20 μg / mL.

[0100] Sample diluent 05: distilled water.

[0101] 3. Carry out automated testing on the computer: The mixed negative sample NM001 and positive sample PM001 were diluted 200 times with the five prepared sample diluents, and automated testing was performed on the machine to evaluate the treatment effects of different diluents.

[0102] 4. Test results: (1) Sample results of IDEXX test kit: Table 4

[0103] (2) Results of treating NM001 and PM001 with different sample dilutions Table 5

[0104] The signal-to-noise ratio of sample dilution 04 is the highest, indicating that the treatment solution prepared by the present invention can more effectively extract the components of Mycobacterium phlei cells, so that it can weaken the cross reaction and improve the specificity of the detection reagent.

[0105] Example 9 Sample dilution optimization 1. Sample collection: The samples used were the two mixed samples in Example 8, the mixed negative sample NM001 and the mixed positive sample PM001.

[0106] 2. Preparation of diluent: The sample dilution solution was prepared according to the following table. The Mycobacterium phlei extract was obtained by treating the bacteria with a homemade treatment solution.

[0107] Table 6

[0108] 3. Automated on-machine processing test: The mixed negative sample NM001 and positive sample PM001 were automatically processed using 12 prepared dilution solutions. The samples were diluted 200 times to evaluate the treatment effects of different dilution solutions.

[0109] 4. Test results: Table 7

[0110] When the salt ion concentration increases to 100mM, the overall light value decreases. The diluent formula 4 and diluent formula 5 have the highest signal-to-noise ratio, both of which can meet the requirements of sample processing. However, considering the cost, diluent 4, that is, a buffer solution with a concentration of 20ug / mL of Mycobacterium phlei extract, 50mM PBS, and 0.9% NaCl, was selected as the sample diluent for the test reagent applied for preparation. In addition, the signal-to-noise ratio of diluent formula 11 and diluent formula 12 is also high, that is, a buffer solution with a concentration of 20-30ug / mL of Mycobacterium phlei extract, a concentration of 40-60mM PBS, and a concentration of 0.8-1.0% NaCl has a better effect as a sample diluent.

[0111] 5. Verification experiment The screened sample processing solution 4 was used to process 20 random samples, and a comparative test was performed using the detection kit prepared in the present application and the IDEXX reagent.

[0112] The kit components include streptavidin magnetic particles (0.4 mg / mL), biotin-labeled bovine paratuberculosis antigen (0.5 μg / mL), acridine sulfonamide-labeled anti-bovine IgG antibody (1 μg / mL), sample diluent (Mycobacterium phlei extract concentration 20 ug / mL, 50 mM PBS, 0.9% NaCl), low-value calibrator, high-value calibrator, negative quality control, and positive quality control. The sample was diluted 200 times, and the diluted solution was automatically tested on the machine. The results are shown in the following table.

[0113] Table 8

[0114] The test kit prepared in this example is positive if the result is ≥120 AU / mL; negative if the result is <90 AU / mL; and gray if the result is between 90-120 AU / mL.

[0115] The above results show that the positive and negative consistency rate of the test kit prepared in the application is 100% with that of IDEXX reagent.

[0116] Example 10 Test kit sensitivity and specificity sample testing The prepared bovine Mycobacterium paratuberculosis antibody detection kit was used to detect 122 samples from Tianjin Customs Animal and Plant and Food Inspection Center, and the samples were compared with IDEXX's Mycobacterium paratuberculosis Avm subspecies antibody detection kit. The positive coincidence rate, negative coincidence rate and total coincidence rate of the reagent to be evaluated and the comparison reagent were statistically analyzed using a four-cell table.

[0117] Table 9

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A sample diluent, characterized in that: The sample diluent includes 20-30ug / mL Mycobacterium phlei extract, 40-60mM PBS and 0.8-1.0% NaCl.

2. The sample diluent according to claim 1, characterized in that: The sample diluent includes 20 ug / mL Mycobacterium phlei extract, 50 mM PBS and 0.9% NaCl.

3. The sample diluent according to any one of claims 1 to 2, characterized in that: The preparation of the Mycobacterium phlei extract comprises the following steps: (1) inoculating Mycobacterium phlei into a fermentation medium for fermentation to obtain a fermentation liquid; (2) The obtained fermentation broth is inactivated with formaldehyde, centrifuged, the bacterial cells are collected, and the treatment liquid is added. After treatment at 80-90° C. for 0.4-1 h, ultrasonication, centrifugation, and filtration are performed to obtain the Mycobacterium phlei extract.

4. The sample diluent according to claim 3, characterized in that: The fermentation medium described in step (1) includes 2-6 g / L soy peptone, 2-6 g / L tryptone, 2-6 g / L yeast extract, 2-6 g / L glucose, 2-6 g / L sucrose, 0.4-0.6 g / L magnesium sulfate, 0.4-0.8 g / L disodium hydrogen phosphate, 0.4-0.8 g / L potassium dihydrogen phosphate, 0.4-0.8 g / L sodium chloride, and 8-12 g / L glycerol.

5. The sample diluent according to claim 3, characterized in that: The fermentation culture in step (1) is carried out under the conditions of 35-38°C, a rotation speed of 120-180 rpm, and a ventilation ratio of 1:0.8-2 for 48-96 hours.

6. The sample diluent according to claim 3, characterized in that: The concentration of formaldehyde in step (2) is 0.2-0.6%; the volume ratio of the fermentation liquid to formaldehyde in step (2) is 1:0.8-2; the mass volume ratio of the wet weight of the bacterial cells to the treated liquid in step (2) is 1:4-6.

7. The sample diluent according to claim 3, characterized in that: The treatment solution in step (2) has a formula of 10-20 mM PBS, 0.5-2 mM EDTA, 40-60 mM sodium glutamate, a final concentration of 0.2-0.4% glycine, 1-2% sucrose, 1-3% trehalose, 1-2% mannitol, 0.05-0.1% Tween-20, 0.2-0.4% poloxamer, 0.4-0.6% CHAPS and 0.1-0.2% SDS, pH 6-8.

8. Use of the sample diluent according to any one of claims 1 to 7 in preparing a bovine paratuberculosis screening kit.

9. A chemiluminescent detection kit for bovine paratuberculosis screening, characterized in that: The chemiluminescent detection kit comprises the sample diluent according to any one of claims 1 to 7.

10. Use of the chemiluminescence detection kit according to claim 9 in screening for bovine paratuberculosis.

Images