A rapid detection kit and method for mastitis of dairy buffaloes

Abstract

The application discloses a rapid detection kit and a detection method for water buffalo mastitis, and relates to the technical field of rapid diagnosis of animal diseases.The kit comprises a somatic cell fluorescent staining reagent module, an immunochromatographic detection card module, a water buffalo mastitis somatic cell counting standard color card, a water buffalo mastitis determination standard control table and a sample pretreatment reagent.The sample pretreatment reagent is used to disperse and remove high-content fat globules in water buffalo milk and lyse red blood cells to eliminate interference, nucleic acid fluorescent dyes are used to stain somatic cells, and the standard color card is used for comparison to realize quantitative detection of somatic cells.Meanwhile, the immunochromatographic detection card is used for rapid identification of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus mastitidis and Escherichia coli, and comprehensive determination is carried out according to the threshold standard of water buffalo somatic cells, so that the infection state screening of water buffalo mastitis can be rapidly and integrally completed on a pasture, and the false positive rate and detection cost are reduced.

Description

Technical Field

[0001] This invention relates to the field of rapid diagnostic technology for animal diseases, specifically to a rapid detection kit and method for mastitis in dairy buffalo. Background Technology

[0002] Buffalo are important dairy livestock, producing milk with high fat content and rich nutritional value, making it a crucial raw material for producing high-quality buffalo milk products. Mastitis is one of the most prevalent and economically damaging diseases in dairy buffalo farming, especially subclinical mastitis, which is difficult to detect due to the lack of visible abnormalities in the milk, resulting in significant hidden economic losses. Therefore, developing a rapid mastitis detection technology suitable for dairy buffalo is of great importance for ensuring farming efficiency and dairy product quality.

[0003] Current mastitis detection technologies lack specific sample pretreatment protocols tailored to the high fat and casein content of dairy cow milk. Fat globules and milk proteins severely interfere with somatic cell fluorescence staining, leading to decreased accuracy and sensitivity. Furthermore, existing products are functionally limited. The California mastitis assay only allows for subjective semi-quantitative screening, and immunochromatographic products detect only single or a few pathogens. They cannot simultaneously determine mastitis infection status, perform semi-quantitative somatic cell concentration, and identify major pathogens in a single test. Moreover, there is a lack of somatic cell threshold standards applicable to dairy cows; directly applying the 200,000 cells / mL threshold for dairy cows results in a high false-positive rate. Precision methods such as flow cytometry and PCR require specialized equipment and personnel, making them unsuitable for on-farm use.

[0004] In summary, existing technologies cannot meet the needs for rapid and comprehensive on-site diagnosis of mastitis in dairy buffalo. There is an urgent need to develop a rapid detection kit and method specifically designed for the characteristics of dairy buffalo milk, capable of simultaneously performing semi-quantitative somatic cell detection and identification of major pathogens. Summary of the Invention

[0005] To address the shortcomings of existing detection technologies for mastitis in dairy buffalo, such as the lack of dedicated sample pretreatment methods, limited detection capabilities, and the inability to rapidly perform comprehensive diagnosis on-site, this invention presents a rapid detection kit and method for mastitis in dairy buffalo. The kit includes a somatic cell fluorescence staining module, an immunochromatographic detection card module, a standard colorimetric card for somatic cell counting in dairy buffalo, a reference table for determining mastitis in dairy buffalo, and sample pretreatment reagents. These reagents disperse and remove high levels of fat globules and lyse red blood cells in dairy buffalo milk to eliminate interference. Nucleic acid fluorescent dyes are used to stain somatic cells, and the results are compared with the standard colorimetric card to achieve quantitative detection of somatic cells. Simultaneously, the immunochromatographic detection card is used to rapidly identify Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus lactis, and Escherichia coli. A comprehensive assessment is then performed according to a dairy buffalo-specific somatic cell threshold standard. This kit enables rapid, integrated screening of mastitis infection status and identification of major pathogens on-site, reducing false positive rates and detection costs, and improving detection accuracy and practicality.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: On the one hand, a rapid detection kit for mastitis in dairy buffalo, the detection kit comprising: a somatic cell fluorescence staining reagent module, an immunochromatographic detection card module, a somatic cell counting standard colorimetric card for mastitis in dairy buffalo, and sample pretreatment reagents; The somatic cell fluorescence staining reagent module includes a nucleic acid fluorescent dye working solution, a fat globule dispersant, and a red blood cell lysis buffer. The nucleic acid fluorescent dye in the working solution is either acridine orange or propidium iodide. The concentration of the nucleic acid fluorescent dye working solution is 5-50 μg / mL, and the solvent of the nucleic acid fluorescent dye working solution is phosphate buffer with pH 7.0-7.4; The fat globule dispersant is a Triton X-100 aqueous solution with a mass concentration of 0.05%-0.2% or a Tween-20 aqueous solution with a mass concentration of 0.01%-0.1%. The erythrocyte lysis buffer is an aqueous solution containing 0.83% ammonium chloride, 0.1% sodium bicarbonate, and 0.01 mM ethylenediaminetetraacetic acid, and the pH value of the erythrocyte lysis buffer is 7.2-7.4. The immunochromatographic detection card module contains five immunochromatographic detection cards for the pathogen of mastitis in dairy buffalo. The immunochromatographic detection card for the pathogen of mastitis in dairy buffalo is a lateral flow immunochromatographic test strip. The lateral flow immunochromatographic test strip includes, from the sample pad end to the absorbent pad end, a sample pad, a conjugation pad, a nitrocellulose membrane, and an absorbent pad in sequence. The somatic cell count standard colorimetric card for bovine mastitis is a colorimetric card printed with different fluorescence intensity gradient patterns. The different fluorescence intensity gradient patterns correspond to the standard fluorescence colorimetric results when the somatic cell concentration in bovine milk is 0 cells / mL, 50,000 cells / mL, 100,000 cells / mL, 200,000 cells / mL, 500,000 cells / mL, 1,000,000 cells / mL, 2,000,000 cells / mL, and 5,000,000 cells / mL, respectively. The sample pretreatment reagent includes a milk diluent and a milk defatting agent; The emulsion diluent is a PBS buffer solution with a pH of 7.2-7.4 containing 0.9% sodium chloride and 0.05% Tween-20. The milk defatting agent is a PBS buffer containing 2%-5% polyethylene glycol octylphenyl ether by mass.

[0007] Furthermore, the test kit also includes auxiliary tools, including a micropipette, disposable sampling tubes, a milk collection cup for dairy buffalo, a timer, and an ultraviolet excitation lamp.

[0008] Furthermore, the conjugate pad is immobilized with a colloidal gold-labeled detection antibody, which is a monoclonal or polyclonal antibody against at least one of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus lactis, and Escherichia coli. The nitrocellulose membrane is provided with a detection line and a control line. The detection line is coated with a capture antibody corresponding to the colloidal gold-labeled detection antibody, and the control line is coated with goat anti-mouse IgG antibody or rabbit anti-mouse IgG antibody.

[0009] Furthermore, the colloidal gold-labeled detection antibody is a colloidal gold-labeled monoclonal antibody against Staphylococcus aureus protein A, the capture antibody on the detection line is a rabbit polyclonal antibody against Staphylococcus aureus protein A, and the particle size of the colloidal gold is 20-60 nm.

[0010] Furthermore, the immunochromatographic detection card module includes five immunochromatographic detection cards for bovine mastitis pathogens, which are respectively targeted at Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus lactis, and Escherichia coli.

[0011] On the other hand, a detection method for a rapid diagnostic kit for mastitis in dairy bovines, the specific steps of which are as follows: S100. Before milking the buffalo, disinfect the teats, discard the milk from the first three milkings, and collect milk samples from each udder area. Take 1 mL of milk sample and add it to 9 mL of milk diluent and mix well to obtain a 10-fold diluted milk sample. Take 1 mL of the 10-fold diluted milk sample and add it to 1 mL of milk defatting reagent, vortex for 30 s, let stand for 5 min, and take the supernatant as the sample solution to be tested. S200. Take 100 μL of the sample solution to be tested and add 10 μL of red blood cell lysis buffer. Mix well and let stand at room temperature for 3 min. Add 5 μL of nucleic acid fluorescent dye working solution, mix well and incubate at room temperature in the dark for 2 min. Place the stained sample solution under a UV excitation lamp to observe the fluorescence intensity and compare it with the somatic cell count standard colorimetric card for bovine mastitis to determine the somatic cell concentration. S300. Take 200 μL of the sample solution to be tested and add it to the sample well of the milk buffalo mastitis pathogen immunochromatographic detection card for pathogen detection. After standing at room temperature for 10-15 minutes, observe the color development of the detection line and the control line. S400. A comprehensive judgment is made based on the somatic cell concentration obtained in step S200 and the pathogen detection results obtained in step S300.

[0012] Furthermore, in step S100, the method for disinfecting the cow's teats is to wipe the teats with a cotton ball soaked in 75% alcohol. In S200, the wavelength of the ultraviolet excitation lamp is 365nm or 450nm, and the nucleic acid fluorescent dye is either acridine orange or propidium iodide.

[0013] Furthermore, in S300, the criteria for judging the results of pathogen detection are as follows: If the control line shows color but the test line does not, the result is considered negative for the corresponding pathogen. If both the control line and the test line show color, it is determined to be positive for the corresponding pathogen. If the control line does not develop color, the test result is deemed invalid and must be retested.

[0014] Furthermore, in S400, the comprehensive judgment criterion is as follows: A cow's milk with a somatic cell count of less than 250,000 cells / mL and negative pathogen tests are considered healthy. A bovine milk somatic cell count ≥250,000 / mL and <500,000 / mL is considered a suspected infection. A bovine milk somatic cell count ≥500,000 / mL is considered a mastitis infection, and the type of pathogen is determined based on the pathogen detection results.

[0015] Compared with existing technologies, this rapid detection kit and method for mastitis in dairy buffalo has the following advantages: I. This invention solves the problem of interference from high milk fat and high casein content in bovine milk on somatic cell fluorescence staining detection by setting up a sample pretreatment module containing a fat globule dispersant and a milk defatting reagent. The fat globule dispersant reduces the surface tension of fat globules, making them uniformly dispersed and preventing them from agglomerating. The Triton X-100 in the milk defatting reagent disrupts the fat globule membrane structure, promoting the separation of fat from whey. After centrifugation or settling, the supernatant is taken, reducing the fat and casein content in the sample solution to ensure that the subsequently added nucleic acid fluorescent dye can fully bind to the DNA in the somatic cell nucleus. The linear relationship between fluorescence signal intensity and somatic cell concentration is guaranteed. The erythrocyte lysis buffer selectively lyses residual erythrocytes in the milk, eliminating the interference of erythrocyte DNA on somatic cell fluorescence counting, avoiding fluorescence quenching and background interference caused by the high fat content of bovine milk, and improving detection sensitivity and specificity.

[0016] II. This invention integrates a somatic cell fluorescence staining quantitative detection module and an immunochromatographic pathogen detection module into the same kit. The somatic cell fluorescence staining module uses the fluorescence intensity generated after acridine orange binds to somatic cell DNA to reflect the number of somatic cells. Semi-quantitative results are obtained by comparing with a standard colorimetric card printed with concentration gradients. The immunochromatographic detection card module adopts the double antibody sandwich principle of colloidal gold-labeled antibody and capture antibody to detect five major pathogens: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus lactis, and Escherichia coli. A single sampling can obtain dual information on infection status and pathogen type, making it suitable for field application in ranches.

[0017] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0019] Figure 1 This is a flowchart of a rapid test kit for mastitis in dairy buffalo. Detailed Implementation

[0020] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0021] In the description of this invention, the specific numerical values ​​appearing in the embodiments, such as concentration, volume, temperature, time, spray volume, overlap length, and judgment threshold, are all exemplary values. Those skilled in the art can make reasonable adjustments according to actual detection conditions and reagent sources, and the above specific values ​​should not be construed as limiting the scope of protection of this invention. The reagents, antibodies, standard strains, instruments, and equipment used in the embodiments, unless otherwise specified, can all be prepared by known methods or obtained from commercial channels. Furthermore, the technical features involved in the different embodiments of this invention described below can be combined with each other as long as they do not conflict with each other. The technical solutions of this invention are further illustrated below through specific embodiments. The embodiments given are used to verify the feasibility and technical effects of the technical solutions of this invention and do not constitute a limitation on the scope of protection of this invention. Example 1

[0022] This embodiment achieves the complete assembly of a rapid detection kit for mastitis in dairy buffalo by preparing a somatic cell fluorescence staining reagent module, an immunochromatographic detection card module, a somatic cell counting standard colorimetric card, sample pretreatment reagents, and auxiliary tools.

[0023] Preparation of somatic cell fluorescence staining reagent module: Preparation of working solution for nucleic acid fluorescent dye: Weigh 5 mg of acridine orange powder and dissolve it in 100 mL of pH 7.2 phosphate buffer to prepare a working solution with a concentration of 50 μg / mL. Filter the solution through a 0.22 μm microporous membrane for sterilization, aliquot it into brown centrifuge tubes, and store it at 4°C in the dark. The shelf life of this working solution is 6 months.

[0024] Preparation of the fat globule dispersant: Take 0.1 mL of Triton X-100 stock solution, add it to 99.9 mL of deionized water, mix thoroughly, and prepare a 0.1% (w / w) Triton X-100 aqueous solution. This fat globule dispersant should be stored at room temperature.

[0025] Preparation of erythrocyte lysis buffer: Weigh 8.29 g of ammonium chloride and 1.0 g of sodium bicarbonate, add 1 mL of 0.01 M ethylenediaminetetraacetic acid (EDTA) solution, and bring the volume to 1000 mL with deionized water. Adjust the pH to 7.3. Filter the buffer through a 0.22 μm microporous membrane for sterilization and store at 4 °C. The mass concentration of ammonium chloride in this erythrocyte lysis buffer is 0.83%, the mass concentration of sodium bicarbonate is 0.1%, and the concentration of EDTA is 0.01 mM.

[0026] Preparation of immunochromatographic detection card modules: Taking the Staphylococcus aureus detection card as an example, the preparation methods of the non-lactating streptococcus detection card, discontinued lactation streptococcus detection card, mammary streptococcus detection card and Escherichia coli detection card are the same as those of the Staphylococcus aureus detection card. The only difference is that the detection antibody and the capture antibody are antibodies against the corresponding pathogens.

[0027] Preparation of colloidal gold-labeled antibodies: A colloidal gold solution with a particle size of 40 nm was prepared using the trisodium citrate reduction method. 100 mL of 0.01% chloroauric acid aqueous solution was heated to boiling. While stirring, 2.5 mL of 1% trisodium citrate aqueous solution was added, and the mixture was boiled for another 15 min. After cooling to room temperature, a wine-red colloidal gold solution was obtained. The pH of the wine-red colloidal gold solution was adjusted to 8.0 using 0.1 M potassium carbonate solution. While stirring, a monoclonal antibody against Staphylococcus aureus protein A was added at a ratio of 0.1 mg antibody per mL of colloidal gold solution. The mixture was stirred for another 30 min, and then 1% bovine serum albumin was added, followed by blocking for 30 min. The mixture was centrifuged at 12000 rpm for 30 min at 4 °C. The supernatant was discarded, and the precipitate was resuspended in phosphate buffer containing 1% bovine serum albumin and 5% sucrose to 1 / 10 of its original volume to obtain the colloidal gold-labeled antibody working solution, which was stored at 4 °C protected from light for later use.

[0028] Coating of the nitrocellulose membrane: Rabbit polyclonal capture antibody against Staphylococcus aureus protein A (concentration 1 mg / mL) was diluted to 0.5 mg / mL with coating buffer, which was phosphate buffer containing 3% methanol. The diluted capture antibody was streaked onto the nitrocellulose membrane at a spray rate of 1 μL / cm as the test line. Goat anti-mouse IgG antibody (concentration 0.5 mg / mL) was streaked onto the same nitrocellulose membrane in the same manner as the control line. The test line and the control line were spaced 4 mm apart. After streaking, the nitrocellulose membrane was dried at 37°C for 2 hours to obtain the coated nitrocellulose membrane.

[0029] Treatment of the conjugate pad: The glass fiber conjugate pad was soaked in phosphate buffer containing 1% bovine serum albumin and 2% sucrose for 30 min, dried at 37°C, and the colloidal gold-labeled antibody working solution was uniformly sprayed onto the conjugate pad at a spray rate of 10 μL / cm. The conjugate pad was then vacuum dried at 37°C for 4 h to obtain the conjugate pad.

[0030] The sample pad, conjugate pad, coated nitrocellulose membrane, and absorbent pad are sequentially glued onto a PVC base plate, with a 2mm overlap between each layer. The assembled test strip is then cut into 4mm wide strips using a strip cutter and placed into plastic cartridges. These cartridges have a sample application hole and an observation window; the sample application hole corresponds to the sample pad position, and the observation window corresponds to the detection line and control line on the nitrocellulose membrane. The assembled test card is sealed with a desiccant and stored at 4°C. The test card has a shelf life of 12 months.

[0031] Preparation of a standard colorimetric card for somatic cell count in dairy buffalo mastitis: Prepare standard dairy buffalo milk samples with somatic cell concentrations of 0 cells / mL, 50,000 cells / mL, 100,000 cells / mL, 200,000 cells / mL, 500,000 cells / mL, 1,000,000 cells / mL, 2,000,000 cells / mL, and 5,000,000 cells / mL.

[0032] The preparation method is as follows: Using healthy buffalo milk as a matrix, add a known number of buffalo peripheral blood leukocytes, and mix thoroughly to obtain standard milk samples. Take 100 μL of each concentration of standard milk sample, add 10 μL of the above-mentioned erythrocyte lysis buffer, mix well, and let stand at room temperature for 3 min. Add 5 μL of acridine orange working solution with a concentration of 50 μg / mL, mix well, and incubate at room temperature in the dark for 2 min. Add the stained sample solution to a glass slide, cover with a coverslip, and observe and photograph under a 365 nm ultraviolet excitation lamp. Arrange the fluorescence images corresponding to different somatic cell concentrations according to the concentration gradient, print them on an opaque card, and label the corresponding somatic cell concentration value below each image to obtain the buffalo mastitis somatic cell count standard colorimetric card.

[0033] Preparation of sample pretreatment reagents: Preparation of the milk diluent: Weigh 9.0 g of sodium chloride, 1.44 g of disodium hydrogen phosphate, 0.24 g of potassium dihydrogen phosphate, and 0.2 g of potassium chloride, and dissolve them in 800 mL of deionized water. Add 0.5 mL of Tween-20, mix thoroughly, adjust the pH to 7.4, and bring the volume to 1000 mL with deionized water. The mass concentration of sodium chloride in this milk diluent is 0.9%, the mass concentration of Tween-20 is 0.05%, and the pH value is 7.4.

[0034] Preparation of milk defatting reagent: Weigh 3.0 g of Triton X-100 and dissolve it in 100 mL of pH 7.4 phosphate buffer. Mix thoroughly. The mass concentration of Triton X-100 in this milk defatting reagent is 3%.

[0035] The auxiliary tools include: Micropipettes: one each with a volume range of 100–1000 μL and 0.5–10 μL; Disposable sampling tubes: sterile centrifuge tubes with a capacity of 15 mL; Milk collection cups for dairy buffalo: sterile sampling cups with a capacity of 500 mL; Timer: electronic timer with a timing accuracy of 1 second; Ultraviolet excitation lamp: handheld ultraviolet lamp with a wavelength of 365 nm.

[0036] All the auxiliary tools mentioned are commercially available products and are packaged together with the somatic cell fluorescence staining reagent module in the kit.

[0037] This embodiment completes the assembly of a rapid detection kit for mastitis in dairy buffalo by preparing somatic cell fluorescence staining reagents, preparing five pathogen immunochromatographic detection cards, making standard colorimetric cards, preparing pretreatment reagents, and equipping auxiliary tools, thus providing a material basis for subsequent on-site testing. Example 2

[0038] Based on Example 1, this embodiment achieves a comprehensive determination of the mastitis infection status of buffalo by pretreatment of the milk sample to be tested, quantitative fluorescence staining, and pathogen detection by immunochromatographic analysis.

[0039] like Figure 1 As shown, the specific steps of the detection method of a rapid test kit for mastitis in dairy buffalo are as follows: S100. Before milking the buffalo, disinfect the teats, discard the milk from the first three milkings, and collect milk samples from each udder area. Take 1 mL of milk sample and add it to 9 mL of milk diluent and mix well to obtain a 10-fold diluted milk sample. Take 1 mL of the 10-fold diluted milk sample and add it to 1 mL of milk defatting reagent, vortex for 30 s, let stand for 5 min, and take the supernatant as the sample solution to be tested. S200. Take 100 μL of the sample solution to be tested and add 10 μL of red blood cell lysis buffer. Mix well and let stand at room temperature for 3 min. Add 5 μL of nucleic acid fluorescent dye working solution, mix well and incubate at room temperature in the dark for 2 min. Place the stained sample solution under a UV excitation lamp to observe the fluorescence intensity and compare it with the somatic cell count standard colorimetric card for bovine mastitis to determine the somatic cell concentration. S300. Take 200 μL of the sample solution to be tested and add it to the sample well of the milk buffalo mastitis pathogen immunochromatographic detection card for pathogen detection. After standing at room temperature for 10-15 minutes, observe the color development of the detection line and the control line. S400. A comprehensive judgment is made based on the somatic cell concentration obtained in step S200 and the pathogen detection results obtained in step S300.

[0040] In this embodiment, three lactating Mora buffaloes from a dairy buffalo farm were selected and numbered A, B, and C. Samples were taken before milking in the morning. Each teat was disinfected with 75% alcohol swabs. After discarding the milk from the first three milkings, 5 mL milk samples were collected from each of the four milking areas using sterile milk collection cups. The milking areas were labeled with numbers: left anterior, right anterior, left posterior, and right posterior.

[0041] Take 1 mL of milk sample from each milk zone and add it to 9 mL of milk diluent. Mix thoroughly to obtain a 10-fold diluted milk sample. Take 1 mL of the above 10-fold diluted milk sample and add it to 1 mL of milk defatting reagent. Vortex for 30 seconds, let stand for 5 minutes, and take the supernatant as the sample solution to be tested.

[0042] Somatic cell fluorescence staining and quantitative detection: Add 100 μL of the sample solution to a microcentrifuge tube, add 10 μL of erythrocyte lysis buffer, mix well and let stand at room temperature for 3 min, add 5 μL of acridine orange working solution with a concentration of 50 μg / mL, mix well and incubate at room temperature in the dark for 2 min, drop the stained sample solution onto a glass slide, cover with a coverslip, and observe the fluorescence under a 365 nm ultraviolet excitation lamp.

[0043] The observed fluorescence intensity was compared with the standard colorimetric chart for somatic cell count in bovine mastitis to determine the somatic cell concentration in milk samples from each udder region.

[0044] Take 200 μL of the sample solution to be tested and add it dropwise into the sample wells of the Staphylococcus aureus test card, Streptococcus agalactiae test card, Streptococcus dysgalactiae test card, Streptococcus lactis test card, and Escherichia coli test card, respectively; after standing at room temperature for 15 min, observe the color development of the test line and control line of each test card.

[0045] If the control line develops color but the test line does not, the result is considered negative for the corresponding pathogen; if both the control line and the test line develop color, the result is considered positive for the corresponding pathogen; if the control line does not develop color, the result is considered invalid and retesting is required.

[0046] Test results Of the 12 samples from the breast milk area, 5 tested positive for Staphylococcus aureus, while the other 4 pathogens were negative. The specific test results are shown in Table 1. Table 1: Test Results of Samples from the Milk Section of Buffalo Milk Area in Example 2

[0047] In Table 1, the unit of somatic cell count is 10,000 cells / mL. Samples labeled B (left rear) represent mastitis caused by Staphylococcus aureus, and samples labeled C (left front) represent mastitis caused by Staphylococcus aureus. For breast samples with positive or suspicious results, the California mastitis assay and flow cytometry somatic cell counting were used for verification. The comparison results are shown in Table 2. Table 2: Comparison of the method of the present invention with the somatic cell counting method by flow cytometry

[0048] In Table 2, "Trace" and "+" are both grading symbols for the interpretation results of the California Mastitis Detection Method (CMT), specifically: Trace indicates microaggregation, meaning that a small amount of flocculent precipitate appears after the milk is mixed with CMT reagent, but the degree of aggregation is not obvious. When the test plate is tilted, a small amount of particulate matter can be seen flowing slowly.

[0049] + indicates a weak positive result, meaning that a significant agglutination reaction occurs when the milk is mixed with CMT reagent, and the agglutinate can be seen flowing when the test plate is tilted, but it has not yet formed a gel-like substance.

[0050] ++ indicates a positive result, with obvious agglutination reaction. The mixture appears gelatinous, and the agglutinate does not flow when the test plate is tilted.

[0051] +++ indicates a strong positive result, with a strong agglutination reaction. The mixture forms a distinct gel-like consistency with a raised surface. When the test plate is tilted, the agglutinate adheres to the bottom of the plate and does not flow.

[0052] The results showed that the deviation between the somatic cell count results obtained by the method of the present invention and the standard flow cytometry method was within 10%. The California mastitis detection method could not provide precise values ​​and could only perform semi-quantitative grading, while the method of the present invention could provide more accurate semi-quantitative somatic cell count results. In this embodiment, by pretreatment of the breast milk samples to be tested, semi-quantitative fluorescence staining, and pathogen detection by immunochromatography, the mastitis infection status and pathogen identification of 12 breast milk samples were completed within 30 minutes.

[0053] Example 3

[0054] This embodiment determines the sensitivity of the immunochromatographic test card and somatic cell fluorescence staining detection by preparing simulated positive samples of different concentrations and standard samples of different somatic cell concentrations, and verifies the specificity of the immunochromatographic test card by detecting multiple pathogens.

[0055] Using healthy cow's milk as a base, and adding known concentrations of Staphylococcus aureus standard strains, preparations were made with bacterial concentrations of [missing information]. CFU / mL CFU / mL CFU / mL CFU / mL For simulated positive samples of CFU / mL, take 200μL of each concentration of simulated positive sample and add them to the sample well of the Staphylococcus aureus test card. After standing at room temperature for 15 minutes, observe the color development of the test line.

[0056] The results showed that when the bacterial concentration was ≥ At CFU / mL, the test line shows clear color development; therefore, the limit of detection for the Staphylococcus aureus immunochromatographic assay card is [limit value missing]. CFU / mL.

[0057] Using healthy cow's milk as a matrix, standard samples were prepared with known numbers of peripheral blood leukocytes, resulting in somatic cell concentrations of 10,000, 20,000, 50,000, 100,000, 200,000, and 500,000 cells / mL. Somatic cell fluorescence staining and quantitative detection were performed according to Example 2, and the results were compared with a standard colorimetric card.

[0058] The results showed that when the somatic cell concentration was ≥50,000 cells / mL, the fluorescence intensity was distinguishable from the blank control. Therefore, the detection limit for somatic cell fluorescence staining is 50,000 cells / mL.

[0059] Common pathogens causing mastitis in dairy buffalo (Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus lactis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) and common non-mastitis bacteria (Lactobacillus, Bacillus) were selected, and concentrations of [missing information] were prepared respectively. CFU / mL bacterial suspensions. Take 200 μL of each bacterial suspension and add it dropwise to the sample well of the Staphylococcus aureus test card. After standing at room temperature for 15 min, observe the color development of the test line.

[0060] The results showed that the Staphylococcus aureus test card only showed a positive reaction for Staphylococcus aureus, and a negative reaction for other pathogens and common non-mastitis bacteria. Therefore, the Staphylococcus aureus immunochromatographic test card has good specificity.

[0061] This embodiment determines the lowest detection limit of the immunochromatographic assay card through sensitivity verification. The detection limit for CFU / mL and somatic cell fluorescence staining is 50,000 cells / mL, and specificity verification has proven that the Staphylococcus aureus test card has no cross-reactivity with common pathogens and common non-pathogenic bacteria.

[0062] Example 4

[0063] This embodiment verifies the detection performance and application value of the reagent kit under actual production conditions by conducting field applications in five dairy buffalo farms.

[0064] The kit of this invention was applied to 860 lactating dairy buffaloes from 5 dairy buffalo farms, and a total of 3440 milk plot samples were tested. The results showed that 2856 milk plots were negative, accounting for 83.0%; 324 milk plots were questionable, accounting for 9.4%; and 260 milk plots were positive, accounting for 7.6%. The pathogen detection results in the positive milk plots are shown in Table 3. Table 3: Distribution of Pathogen Detection Results in Positive Mammary Zones

[0065] 120 samples from the mammary gland area were simultaneously sent to the laboratory for flow cytometry somatic cell counting and bacterial culture identification. With a flow cytometry somatic cell count ≥500,000 / mL and a positive bacterial culture as the reference standard, the sensitivity of the method of this invention was 94.2%, the specificity was 91.7%, the total accuracy was 93.3%, and the Kappa value was 0.86.

[0066] This embodiment verifies through large-scale field application that the reagent kit of the present invention achieves a sensitivity, specificity, and overall accuracy of over 90% under actual production conditions, and is highly consistent with laboratory standard methods, demonstrating good value for field applications.

[0067] The above embodiments demonstrate that the rapid detection kit and method for mastitis in dairy buffalo provided by the present invention eliminates the interference of high milk fat in dairy buffalo through milk defatting and fat globule dispersion steps. The accuracy of detection is improved by establishing dairy buffalo-specific somatic cell thresholds of 250,000 cells / mL and 500,000 cells / mL. The simultaneous determination of mastitis infection status and pathogen type is achieved through the combined detection of quantitative somatic cell fluorescence staining and pathogen identification by immunochromatographic analysis. The kit and method are simple to operate, rapid in detection, and suitable for dairy buffalo farms and grassroots veterinary stations.

[0068] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A rapid test kit for mastitis in dairy buffalo, characterized in that, The test kit includes: a somatic cell fluorescence staining reagent module, an immunochromatographic detection card module, a standard colorimetric card for somatic cell counting in dairy buffalo mastitis, and sample pretreatment reagents; The somatic cell fluorescence staining reagent module includes a nucleic acid fluorescent dye working solution, a fat globule dispersant, and a red blood cell lysis buffer. The nucleic acid fluorescent dye in the working solution is either acridine orange or propidium iodide. The concentration of the nucleic acid fluorescent dye working solution is 5-50 μg / mL, and the solvent of the nucleic acid fluorescent dye working solution is phosphate buffer with pH 7.0-7.4; The fat globule dispersant is a Triton X-100 aqueous solution with a mass concentration of 0.05%-0.2% or a Tween-20 aqueous solution with a mass concentration of 0.01%-0.1%. The erythrocyte lysis buffer is an aqueous solution containing 0.83% ammonium chloride, 0.1% sodium bicarbonate, and 0.01 mM ethylenediaminetetraacetic acid, and the pH value of the erythrocyte lysis buffer is 7.2-7.

4. The immunochromatographic detection card module contains five immunochromatographic detection cards for the pathogen of mastitis in dairy buffalo. The immunochromatographic detection card for the pathogen of mastitis in dairy buffalo is a lateral flow immunochromatographic test strip. The lateral flow immunochromatographic test strip includes, from the sample pad end to the absorbent pad end, a sample pad, a conjugation pad, a nitrocellulose membrane, and an absorbent pad in sequence. The somatic cell count standard colorimetric card for dairy buffalo mastitis is a colorimetric card printed with patterns of different fluorescence intensity gradients. The sample pretreatment reagent includes a milk diluent and a milk defatting agent; The emulsion diluent is a PBS buffer solution with a pH of 7.2-7.4 containing 0.9% sodium chloride and 0.05% Tween-20. The milk defatting agent is a PBS buffer containing 2%-5% polyethylene glycol octylphenyl ether by mass.

2. The rapid detection kit for mastitis in dairy buffalo according to claim 1, characterized in that, The test kit also includes auxiliary tools, including a micropipette, disposable sampling tubes, a milk collection cup for dairy buffalo, a timer, and an ultraviolet excitation lamp.

3. The rapid detection kit for mastitis in dairy buffalo according to claim 1, characterized in that, The conjugate pad is immobilized with a colloidal gold-labeled detection antibody, which is a monoclonal or polyclonal antibody against at least one of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus lactis, and Escherichia coli. The nitrocellulose membrane is provided with a detection line and a control line. The detection line is coated with a capture antibody corresponding to the colloidal gold-labeled detection antibody, and the control line is coated with goat anti-mouse IgG antibody or rabbit anti-mouse IgG antibody.

4. The rapid detection kit for mastitis in dairy buffalo according to claim 3, characterized in that, The colloidal gold-labeled detection antibody is a colloidal gold-labeled monoclonal antibody against Staphylococcus aureus protein A, and the capture antibody on the detection line is a rabbit polyclonal antibody against Staphylococcus aureus protein A. The particle size of the colloidal gold is 20-60 nm.

5. The rapid detection kit for mastitis in dairy buffalo according to claim 1, characterized in that, The immunochromatographic detection card module includes five immunochromatographic detection cards for bovine mastitis pathogens, which are respectively targeted at Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus lactis, and Escherichia coli.

6. A detection method for a rapid test kit for mastitis in dairy buffalo, applicable to the rapid test kit for mastitis in dairy buffalo as described in any one of claims 1-5, characterized in that, The specific steps of this detection method are as follows: S100. Before milking the buffalo, disinfect the teats, discard the milk from the first three milkings, and collect milk samples from each udder area. Take 1 mL of milk sample and add it to 9 mL of milk diluent and mix well to obtain a 10-fold diluted milk sample. Take 1 mL of the 10-fold diluted milk sample and add it to 1 mL of milk defatting reagent, vortex for 30 s, let stand for 5 min, and take the supernatant as the sample solution to be tested. S200. Take 100 μL of the sample solution to be tested and add 10 μL of red blood cell lysis buffer. Mix well and let stand at room temperature for 3 min. Add 5 μL of nucleic acid fluorescent dye working solution, mix well and incubate at room temperature in the dark for 2 min. Place the stained sample solution under a UV excitation lamp to observe the fluorescence intensity and compare it with the somatic cell count standard colorimetric card for bovine mastitis to determine the somatic cell concentration. S300. Take 200 μL of the sample solution to be tested and add it to the sample well of the milk buffalo mastitis pathogen immunochromatographic detection card for pathogen detection. After standing at room temperature for 10-15 minutes, observe the color development of the detection line and the control line. S400. A comprehensive judgment is made based on the somatic cell concentration obtained in step S200 and the pathogen detection results obtained in step S300.

7. The detection method of the rapid detection kit for mastitis in dairy buffalo according to claim 6, characterized in that, In step S100, the method for disinfecting the teats of dairy cows is to wipe the teats with cotton balls soaked in 75% alcohol. In S200, the wavelength of the ultraviolet excitation lamp is 365nm or 450nm, and the nucleic acid fluorescent dye is either acridine orange or propidium iodide.

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