A borrelia burgdorferi rapid detection kit and detection method

Abstract

The present application belongs to the field of health quarantine, and relates to a borrelia burgdorferi rapid detection kit and a detection method. The kit can perform on-site rapid quantitative detection on a sample to be detected based on fluorescence microsphere immunochromatography technology. The kit comprises a borrelia burgdorferi fluorescence microsphere immunochromatography test strip. The test strip comprises a viscous bottom liner and viscous bottom plate, and is sequentially pasted with an analysis membrane, a binding pad, a water absorption pad and a sample pad. The analysis membrane is a nitrocellulose membrane, which has a detection band and a quality control band, and is respectively fixed with borrelia burgdorferi antigen protein and rabbit anti-SPA. The binding pad is fixed with an SPA and fluorescence microsphere labeled compound. The present application can realize on-site rapid quantitative detection of borrelia burgdorferi carried by mice, and has the advantages of simple operation, short detection time, high detection sensitivity, good specificity, convenient storage and easy popularization and use.

Description

Technical Field

[0001] This invention belongs to the field of health and quarantine, and relates to a rapid detection kit and detection method for Borrelia burgdorferi. The kit is based on fluorescent microsphere immunochromatography technology and can perform rapid on-site quantitative detection of rodent samples. Background Technology

[0002] Vector-borne diseases are a significant class of infectious diseases, accounting for approximately 5%–10% of all infectious disease cases annually in my country, and contributing 30%–40% of all infectious disease deaths. With the development of international trade and tourism, and changes in global climate conditions, the spread of vector-borne diseases is no longer limited by national borders or geographical regions. For example, vector-borne infectious diseases that were previously only found in tropical regions are now frequently appearing in subtropical areas. Rodents are among the most common disease vectors on vehicles entering and leaving border crossings, capable of transmitting over 100 infectious diseases, including scrub typhus, plague, and hemorrhagic fever with renal syndrome, posing a threat to human health. Rodents can also transmit diseases to poultry, livestock, or other wild animals, endangering livestock production.

[0003] Lyme disease is a zoonotic, zoonotic infectious disease caused by *Borrelia burgdorferi*. It is a systemic infection that can cause damage to multiple systems and organs in the body, leading to lifelong disability or even death in severe cases. Clinical symptoms mainly include erythema migraine, multiple erythema radiata, facial nerve palsy, and meningitis. The disease is globally distributed, spreads rapidly, and has a high rate of disability. In recent years, the incidence and epicenter of the disease have shown a rapid upward trend and spread worldwide. According to data from the US CDC, an average of nearly 20,000 new cases occur annually in recent years. Reports estimate that more than 50,000 cases of Lyme disease are diagnosed annually in Europe. With changing travel habits and increasingly frequent cross-border flows of people and goods, the risk of Lyme disease occurrence and transmission in border areas remains high, and the prevention and control of Lyme disease remains a challenging task.

[0004] Currently, methods for detecting Borrelia burgdorferi generally include microscopic examination, pathogen isolation and culture, serological testing (such as enzyme immunoassay), and nucleic acid testing. Microscopic examination is relatively simple to perform, but it requires a high level of professional technical knowledge and practical experience from the testing personnel, making it unsuitable for use at the port of entry. Other testing techniques generally require fixed laboratory facilities and professional technicians, have long testing cycles, and cannot meet the rapid on-site testing needs at ports of entry.

[0005] Fluorescent microspheres generally refer to microspheres whose surface is labeled with fluorescent substances (including surface coating) or whose internal structure contains fluorescent substances (such as embedded or polymerized substances), and which can be excited to fluoresce when stimulated by external energy. Fluorescent microspheres are functional microspheres carrying fluorescent molecules, and their shape can be arbitrary, but they are generally spherical. In recent years, with the deepening research of fluorescent probe technology, it has become possible to prepare fluorescent microspheres with particle sizes ranging from nanometer to submicrometer. Functional microspheres are widely used due to their unique advantages such as large specific surface area, strong surface adsorption and reactivity, and large aggregation effect, as well as the various properties endowed by people according to different applications. Among them, fluorescent microspheres, as a special type of functional microsphere, are increasingly welcomed by researchers due to their stable morphology and structure and stable and efficient luminescence. Newly developed fluorescent microspheres have uniform particle size, good monodispersity, good stability, and high luminescence efficiency. The surface curvature of the microspheres is conducive to the optimal reaction state of the exposed surface of antigen determinants and antibody binding sites, thus finding applications in high-tech fields such as labeling, tracing, immobilized enzymes, and high-throughput drug screening. Polymer microspheres can be used as detection reagents in immunoassays. Microspheres act as carriers, binding to antibodies or antigens through physical adsorption or covalent bonding. Agglutination assays can then be used to detect the corresponding antigens or antibodies in bodily fluids. This method is simple and sensitive, and therefore has broad application prospects.

[0006] Fluorescent microsphere immunochromatography has enabled the advancement of on-site detection technology from qualitative (colloidal gold technology) to quantitative methods, facilitating rapid screening and risk assessment of infectious diseases. Compared to traditional on-site detection techniques (colloidal gold technology), its sensitivity is significantly improved, making it more suitable for detecting vector organisms with low pathogen concentrations. This technology is one of the most advanced rapid on-site detection technologies currently available internationally. Summary of the Invention

[0007] This invention provides a rapid detection kit for Borrelia burgdorferi, enabling rapid on-site quantitative detection of Borrelia burgdorferi in rodents. Another objective of this invention is to provide a method for preparing and applying this rapid detection kit.

[0008] The technical solution adopted by the present invention to achieve the above-mentioned objective is: a rapid detection kit for Borrelia burgdorferi, the kit comprising a Borrelia burgdorferi fluorescent microsphere immunochromatographic test strip, the test strip comprising an analytical membrane, a conjugate pad, an absorbent pad, and a sample pad sequentially pasted on an adhesive backing and an adhesive base plate, the analytical membrane being a nitrocellulose membrane having a detection band and a control band thereon, which respectively immobilize Borrelia burgdorferi antigen protein and rabbit anti-SPA, the conjugate pad immobilizing a SPA-fluorescent microsphere labeled complex.

[0009] The analytical membrane is adhered to the adhesive substrate. One end of the analytical membrane is attached to a binding pad and the other end of the absorbent pad. The sample pad is attached to the binding pad. The other ends of the binding pad and the absorbent pad are aligned with the ends of the adhesive substrate.

[0010] The concentration of Borrelia burgdorferi antigen on the detection band was 1.0 mg / ml, the concentration of rabbit anti-SPA on the control band was 1.6 mg / ml, and the spray volume was 1.2 µl / cm.

[0011] The labeled complex and the labeling complex on the conjugate pad were both applied to the conjugate pad using a spray film instrument at a spray volume of 6 μL / cm and then vacuum dried for 3 hours.

[0012] In the preparation of the Borrelia burgdorferi antigen and fluorescent microsphere labeling complex, the ratio of protein to fluorescent microspheres is as follows: 5 ml of the labeling system contains 5 mg of fluorescent microspheres and 80 µg of Borrelia burgdorferi antigen.

[0013] During the use of the test strip, a sample processing liquid is included. The sample processing liquid consists of a 0.01M pH 7 PB buffer solution containing 1% BSA, 0.05% SDS, and 0.05% Triton X-100.

[0014] The test strip has high detection sensitivity, a detection limit of 1 µg / ml, and good repeatability and stability.

[0015] The preparation method of the rapid detection kit for Borrelia burgdorferi includes the following steps: S1. Preparation of the conjugate pad: A mixture of labeled SPA and fluorescent microspheres was sprayed onto glass fibers as a binding pad. The spray volume was [missing information]. 6µl / cm, freeze-dry for 3 hours, then store in a drying cabinet at 2-8℃ for later use; S2. Analytical membrane preparation: A Millipore medium-speed membrane was used, and the protein solution was diluted with 0.01M pH 7.2 PB buffer solution to detect antigen-containing proteins. The white concentration was 1.0 mg / ml, the quality control rabbit anti-SPA concentration was 1.6 mg / ml, the spraying amount was 1.2 ul / cm, after spraying the film, it was placed in an oven at 37℃ and dried for 1 hour, and then stored in a drying cabinet at 2-8℃ for later use. S3. Test strip assembly: Assemble the analytical membrane, binding pad, sample pad, and absorbent pad sequentially on a fluorescent adhesive substrate, and place it on the gate. Cut the test strips into 4mm wide strips using a strip cutter, assemble them into plastic test strip holders, and store them in a drying cabinet at 2-8℃ for later use.

[0016] The preparation method of the labeled mixture in step S1: The fluorescent microspheres are carboxylated europium fluorescent microspheres with a diameter of 200nm, the microspheres were activated with EDC and NHS before labeling. The specific steps are as follows: Take 50 µl of 1% fluorescent microspheres, add 100 µl of pH 6.0 PB buffer solution, centrifuge at 12000 rpm for 30 min, and discard the supernatant; Add 100 µl of pH 6.0 PB buffer solution, wash once more, centrifuge, and discard the supernatant; Reconstitute with 100 µl PB buffer solution, add 15 µl each of 0.001M EDC and NHS, activate for 1 h, centrifuge at 12000 rpm for 30 min, discard the supernatant, dissolve the microspheres in 5 ml pH 6.0 PB buffer solution, add 80 µg SPA protein, stir at room temperature for 2 h, add 1% BSA-PEG and continue stirring to block for 30 min; Centrifuge at 12000 rpm for 30 min, discard the supernatant, dissolve the precipitate in 100 µL PB buffer solution, and store at 4℃ for later use.

[0017] The application of the described rapid detection kit for Borrelia burgdorferi involves using the kit to achieve on-site, specific, rapid, and quantitative detection of Borrelia burgdorferi in euthanized rodent samples. The specific method includes the following steps: S1. Preparation of sample processing solution: The sample processing solution consists of 0.01M pH 7 PB buffer solution, containing 1% BSA, 0.05% SDS, and... 0.05% Triton X-100; the sample processing solution should be stored in a 4°C freezer for later use, and for long-term storage, it should be frozen at -20°C. S2. Sample Testing: Cut a 1cm x 1cm strip of blood-stained test paper, add 500µL of PBS, mix well, and then take 25µL of the mixture to process with 75µL of sample. Mix the liquid; add it to the sample pad in the sample well of the test strip, and detect it through a fluorescence immunochromatographic reader after 15 minutes. Judge the test result by the T / C value, or use ultraviolet light to observe the color development of the test band and the control band to judge the test result.

[0018] This invention utilizes fluorescent microsphere immunochromatography, employing fluorescent microspheres as carriers and leveraging the ability of Staphylococcus aureus surface proteins to capture mammalian IgG, to establish an indirect fluorescent microsphere immunochromatographic detection mode. This enables rapid, on-site quantitative detection of Borrelia burgdorferi in rodents. The procedure is simple, with a short detection time of only 20 minutes, and results are available 15 minutes after sample addition. The detection sensitivity is 1 µg / mL, and there is no cross-reactivity with hantavirus-positive, bocavirus-positive, or pathogenic leptospirosis-positive samples. The intra- and inter-batch repeatability coefficient of variation is less than 5%. This invention facilitates rapid, on-site quantitative detection of rodent samples. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the structure of the immunochromatographic test strip for the fluorescent microspheres of Borrelia burgdorferi of the present invention.

[0020] Figure 2 The results show the sensitivity evaluation of the immunochromatographic test strip for the fluorescent microspheres of Borrelia burgdorferi of this invention. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to the specific embodiments.

[0022] Example 1

[0023] A rapid detection kit for Borrelia burgdorferi, the kit comprising, as follows Figure 1 The illustrated borophorus fluorescent microsphere immunochromatographic test strip includes an analytical membrane, a conjugate pad, an absorbent pad, and a sample pad sequentially adhered to an adhesive backing and an adhesive base plate. The analytical membrane is a nitrocellulose membrane with a detection band and a control band, which are respectively immobilized with borophorus antigen protein and rabbit anti-SPA. The conjugate pad is immobilized with a SPA-fluorescent microsphere-labeled complex.

[0024] The analytical membrane is adhered to the adhesive substrate. One end of the analytical membrane is attached to a binding pad and the other end of the absorbent pad. The sample pad is attached to the binding pad. The other ends of the binding pad and the absorbent pad are aligned with the ends of the adhesive substrate.

[0025] The concentration of Borrelia burgdorferi antigen on the detection band was 1.0 mg / ml, the concentration of rabbit IgG on the control band was 1.6 mg / ml, and the spray volume was 1.2 µl / cm.

[0026] The labeled complex and the labeling complex on the conjugate pad were both applied to the conjugate pad using a spray film instrument at a spray volume of 6.0 μL / cm and then vacuum dried for 3 hours.

[0027] In the preparation of the SPA-fluorescent microsphere labeling complex, the ratio of protein to fluorescent microspheres is as follows: 5 ml labeling system, 5 mg of fluorescent microspheres, and 80 µg of SPA.

[0028] During the use of the test strip, a sample processing liquid is included. The sample processing liquid consists of a 0.01M pH 7 PB buffer solution containing 1% BSA, 0.05% SDS, and 0.05% Triton X-100.

[0029] The test strip has high detection sensitivity, a detection limit of 1 μg / ml, and good repeatability and stability.

[0030] Example 2

[0031] The preparation method of the rapid detection kit for Borrelia burgdorferi in Example 1 includes the following steps: S1. Preparation of the conjugate pad: Take a mixture of labeled SPA and fluorescent microspheres, spray it onto glass fiber as a binding pad, the spray volume is 6µl / cm, freeze dry for 3h, and store in a desiccator at 2-8℃ for later use; The preparation method of the labeled mixture is as follows: The fluorescent microspheres are carboxylated europium fluorescent microspheres with a diameter of 200 nm. Before labeling, the microspheres are activated with EDC and NHS. The specific steps are as follows: Take 50 µl of 1% fluorescent microspheres, add 100 µl of pH 6.0 PB buffer solution, centrifuge at 12000 rpm for 30 min, and discard the supernatant; Add 100 µl of pH 6.0 PB buffer solution, wash once more, centrifuge, and discard the supernatant; Reconstitute with 100 μL PB buffer solution, add 15 µL each of 0.001M EDC and NHS, activate for 1 h, centrifuge at 12000 rpm for 30 min, discard the supernatant, dissolve the microspheres in 5 mL pH 6.0 PB buffer solution, add 120 µg labeled antibody-Borrelia burgdorferi antibody 2, stir at room temperature for 2 h, add 1% BSA-PEG and continue stirring to block for 30 min; Centrifuge at 12000 rpm for 30 min, discard the supernatant, dissolve the precipitate in 100 µL of PB buffer solution, and store at 4 °C for later use. S2. Analytical membrane preparation: Millipore medium-speed membrane was used. The protein solution was diluted with 0.01M pH7.2 PB buffer solution. The detection concentration of antigen protein was 1.0 mg / ml, the quality control concentration of rabbit anti-SPA was 1.6 mg / ml, the membrane spraying volume was 1.2 μl / cm, and after spraying, it was placed in a 37℃ oven for 1 hour and then stored in a 2-8℃ desiccant for later use. S3. Test strip assembly: Assemble the analytical membrane, binding pad, sample pad, and absorbent pad sequentially on a fluorescent adhesive substrate, and place it on the gate. The test strips are cut into 4mm wide strips on the strip cutting machine, assembled into plastic test strip housings, and stored in a drying cabinet at 2-8℃ for later use.

[0032] Example 3

[0033] The application of the rapid detection kit for Borrelia burgdorferi in Example 1 demonstrates how the kit can be used for on-site, specific, rapid, and quantitative detection of Borrelia burgdorferi in euthanized rodent samples. The specific method includes the following steps: S1. Preparation of sample processing solution: The sample processing solution consists of 0.01M pH 7 PB buffer solution, containing 1% BSA, 0.05% SDS, and... 0.05% Triton X-100; the sample processing solution should be stored in a 4°C freezer for later use, and for long-term storage, it should be frozen at -20°C. S2. Sample Testing: Cut a 1cm x 1cm strip of blood-stained test paper, add 500µL of PBS, mix well, and then take 25µL of the mixture to process with 75µL of sample. Mix the liquid; add it to the sample pad in the sample well of the test strip, and detect it through a fluorescence immunochromatographic reader after 15 minutes. Judge the test result by the T / C value, or use ultraviolet light to observe the color development of the test band and the control band to judge the test result.

[0034] Example 4

[0035] Performance evaluation results of the rapid detection kit for Borrelia burgdorferi in Example 1: 1) Sensitivity evaluation results The Borrelia burgdorferi antigen protein was serially diluted with PBS buffer and tested. The mean of the test results (T / C value) was calculated, and a quantitative curve was plotted using Excel software, with the logarithm of concentration on the ordinate and the logarithm of the T / C value on the abscissa, to show the relationship between antibody concentration and sample detection value. The detection limit of this kit was 1 μg / ml, and the R² of the linear relationship curve was 0.82. The results are as follows. Figure 2 As shown.

[0036] 2) Repeatability evaluation results Twenty immunochromatographic test strips were randomly selected from each batch of preparation, with 10 strips per group, and positive and negative samples were tested separately. The coefficient of variation for positive results was <5%.

[0037] 3) Specificity evaluation results The prepared test strips were used to test samples positive for Hantavirus, Bocavirus, pathogenic Leptospira, and anaplasmic phagocytophilia. The results showed that all of these pathogens were negative, indicating good specificity.

Claims

1. A rapid detection kit for Borrelia burgdorferi, characterized in that, The kit includes a borophorus fluorescent microsphere immunochromatographic test strip. The test strip includes an adhesive backing and an analytical membrane, a conjugate pad, an absorbent pad, and a sample pad adhered to the adhesive backing. The analytical membrane is a nitrocellulose membrane with a detection band and a control band. The detection band and the control band are respectively immobilized with borophorus antigen protein and rabbit anti-SPA. The conjugate pad is immobilized with a SPA-fluorescent microsphere-labeled complex.

2. The rapid detection kit for Borrelia burgdorferi according to claim 1, characterized in that, The analytical membrane is adhered to an adhesive substrate. One end of the analytical membrane is attached to a bonding pad and the other end of the absorbent pad. The other ends of the bonding pad and the absorbent pad are aligned with the ends of the adhesive substrate. A sample pad is attached to the bonding pad.

3. The rapid detection kit for Borrelia burgdorferi according to claim 1, characterized in that, The concentration of Borrelia burgdorferi antigen protein on the detection band was 1 mg / ml, the concentration of rabbit anti-SPA on the quality control band was 1.6 mg / ml, and the spraying volume was 1.2 µl / cm.

4. The rapid detection kit for Borrelia burgdorferi according to claim 1, characterized in that, The labeled complexes on the conjugate pads were all applied to the conjugate pads using a spray film instrument at a spray volume of 6 μL / cm, and then vacuum dried for 3 hours.

5. The rapid detection kit for Borrelia burgdorferi according to claim 1, characterized in that, In the preparation of the labeled complex, the ratio of protein to fluorescent microspheres is as follows: 5 ml of the labeling system contains 5 mg of fluorescent microspheres and 80 µg of SPA.

6. A rapid detection kit for Borrelia burgdorferi according to any one of claims 1 to 5, characterized in that, During the use of the test strip, a sample processing liquid is included. The sample processing liquid consists of a 0.01M pH7 PB buffer solution containing 1% BSA, 0.05% SDS, and 0.05% Triton X-100.

7. A rapid detection kit for Borrelia burgdorferi according to any one of claims 1 to 5, characterized in that, The test strip has good detection sensitivity, with a detection limit of 1 μg / ml, and good repeatability and stability.

8. The method for preparing the rapid detection kit for Borrelia burgdorferi according to claim 1, characterized in that, Includes the following steps: S1. Preparation of the conjugate pad: Take a mixture of labeled SPA and fluorescent microspheres, spray it onto glass fiber as a binding pad, the spray volume is 6µl / cm, freeze dry for 3h, and store in a desiccator at 2-8℃ for later use; S2. Analytical membrane preparation: Millipore medium-speed membrane was used. The protein solution was diluted with 0.01M pH7.2 PB buffer solution. The detection concentration of antigen protein was 1.0 mg / ml, the quality control concentration of rabbit anti-SPA was 1.6 mg / ml, the membrane spraying volume was 1.2 μl / cm, and after spraying, it was placed in a 37℃ oven for 1 hour and then stored in a 2-8℃ desiccant for later use. S3. Test strip assembly: On a fluorescent adhesive backing, assemble the analytical membrane, conjugate pad, sample pad, and absorbent pad in sequence. Place them on a guillotine strip cutter to cut them into 4mm wide test strips, assemble them in a test strip plastic cartridge, and store them in a 2-8℃ drying cabinet for later use.

9. The method for preparing the rapid detection kit for Borrelia burgdorferi according to claim 8, characterized in that, The preparation method of the labeled mixture in step S1 is as follows: The fluorescent microspheres are carboxylated europium fluorescent microspheres with a diameter of 200 nm. Before labeling, the microspheres are activated with EDC and NHS. The specific steps are as follows: Take 50 µl of 1% fluorescent microspheres, add 100 µl of pH 6.0 PB buffer solution, centrifuge at 12000 rpm for 30 min, and discard the supernatant; Add 100 µl of pH 6.0 PB buffer solution, wash once more, centrifuge, and discard the supernatant; Reconstitute with 100 µl of PB buffer solution, add 15 µl each of 0.001M EDC and NHS, activate for 1 h, centrifuge at 12000 rpm for 30 min, discard the supernatant, dissolve the microspheres in 5 ml of pH 6.0 PB buffer solution, add 80 µg of SPA protein, stir at room temperature for 2 h, add 1% BSA-PEG and continue stirring to block for 30 min; Centrifuge at 12000 rpm for 30 min, discard the supernatant, dissolve the precipitate in 100 µL PB buffer solution, and store at 4℃ for later use.

10. The application of the rapid detection kit for Borrelia burgdorferi as described in claim 1, characterized in that, The kit is used to achieve rapid, specific, and quantitative on-site detection of Borrelia burgdorferi in euthanized rodent samples. The specific method includes the following steps: S1. Preparation of sample processing solution: The sample processing solution consists of 0.01M pH7 PB buffer solution containing 1% BSA, 0.05% SDS, and 0.05% Triton X-100. The sample processing solution is stored at 4℃ for later use, and for long-term storage, it is frozen at -20℃. S2. Sample Testing: Cut a 1cm x 1cm strip of blood-stained test paper, add 500µL of PBS, mix well, and then take out 25µL of the mixture and mix it with 75µL of sample processing solution. Add the mixture to the sample pad in the sample well of the test paper. After 15 minutes, detect the result using a fluorescence immunochromatographic reader. The result can be determined by the T / C value, or by observing the color development of the test band and control band under UV light.

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