A helicobacter pylori rapid detection kit and an application method thereof
By combining a composite lysis system, a matrix interference blocking system, and an antigen pre-enriched particle suspension, the problems of insufficient efficiency in lysing the cell walls of Gram-negative bacteria and insufficient inhibition of interfering substances in feces in existing technologies have been solved, achieving high sensitivity and accuracy in the detection of Helicobacter pylori and reducing the false negative rate.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- BEIJING RAINBOW BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-04-03
- Publication Date
- 2026-05-29
AI Technical Summary
Existing Helicobacter pylori fecal antigen detection kits have insufficient efficiency in lysing the cell walls of Gram-negative bacteria in the sample processing solution, insufficient inhibition of interfering substances in feces, difficulty in effectively enriching low-abundance antigens, and lack of sample quality monitoring, resulting in a high false negative rate.
A composite lysis system and a matrix interference blocking system were employed, combined with antigen pre-enriched particle suspension and a dual-line quality control mechanism. The activity of endogenous enzymes in bacteria was activated by simulating physiological stress through a weakly acidic buffer solution, cell structure was destroyed by a combination of surfactants, and the antigen was enriched by magnetic nanoparticles. A dual-line quality control line was set up to monitor sample quality.
It significantly improves the sensitivity and accuracy of Helicobacter pylori detection, reduces the false negative rate, enhances the ability to identify low-abundance antigens, and ensures the reliability and operability of the test results.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biological detection technology, and more specifically, to a rapid detection kit for Helicobacter pylori and its application method. Background Technology
[0002] Helicobacter pylori (HP) is a Gram-negative, spiral-shaped pathogen that colonizes the gastric mucosa. Epidemiological studies show that more than 50% of the global population is infected with this bacterium, and it is a major pathogenic factor for chronic gastritis, peptic ulcers, and gastric cancer. Therefore, early, rapid, and accurate detection of Helicobacter pylori infection is of great significance for the prevention and treatment of digestive tract diseases.
[0003] Currently, methods for detecting Helicobacter pylori mainly include invasive testing (such as gastroscopy biopsy combined with a rapid urease test) and non-invasive testing (such as fecal antigen testing and serological testing). Among these, fecal antigen testing has become one of the preferred methods for large-scale clinical screening and home self-testing due to its advantages such as being non-invasive, simple, and requiring no expensive equipment. This technology typically uses the principle of double-antibody sandwich immunochromatography to detect Helicobacter pylori antigens in fecal samples using specific antibodies.
[0004] However, existing Helicobacter pylori fecal antigen detection kits still have the following technical shortcomings in practical applications: First, the sample processing solution is not efficient enough at lysing the cell walls of Gram-negative bacteria. Helicobacter pylori has a complex double-membrane structure: the outer membrane is maintained stable by "ion bridges" formed by divalent cations, and the inner membrane is a phospholipid bilayer. Existing processing solutions mostly use a single surfactant, which is difficult to penetrate the outer membrane barrier and destroy the inner membrane structure simultaneously, resulting in insufficient antigen release and a high risk of false negatives in low-load infections. Second, the inhibition of interfering substances in fecal samples is insufficient. Feces contain various interfering substances such as bile salts, heterophile antibodies, and free divalent cations, which can easily non-specifically adsorb labeled antibodies, leading to false positives or consuming the active ingredients of the processing solution. Existing technologies mostly use single animal IgG to block heterophile antibodies, but lack effective treatment for the large number of divalent cation interfering substances present in feces. Third, there is a lack of effective monitoring mechanisms for sample quality. Existing kits only have a single quality control line (C line), which can only verify the effectiveness of the test strip and cannot identify "invalid samples" caused by insufficient sampling or sample degradation. Based on the above statements, the present invention provides a rapid detection kit for Helicobacter pylori and its application method. Summary of the Invention
[0005] To address the problems in existing technologies, such as insufficient antigen release due to the lack of specificity of sample processing solutions in lysing the cell walls of Gram-negative bacteria, inadequate inhibition of interfering substances in feces, difficulty in effectively enriching low-abundance antigens, and high false-negative rates due to the lack of effective monitoring of sample quality, this invention provides a rapid detection kit for Helicobacter pylori and its application method.
[0006] In a first aspect, the present invention provides a rapid detection kit for Helicobacter pylori, which adopts the following technical solution: A rapid detection kit for Helicobacter pylori includes a sample processing solution, an antigen pre-enriched particle suspension, and an immunochromatographic test strip. The sample processing solution comprises the following raw materials in parts by weight: 80-95 parts of antigen-induced release system, 2-8 parts of complex lysis system, 1-5 parts of matrix interference blocking system, and 2-3 parts of chromatography promoter; The antigen pre-enriched particles in the antigen pre-enriched particle suspension are magnetic nanoparticles with anti-Helicobacter pylori antibodies coupled to their surface. The immunochromatographic test strip includes a sample pad, a conjugation pad, a nitrocellulose membrane, and an absorbent pad. The nitrocellulose membrane has a first detection line, a second detection line, and a control line arranged sequentially along the chromatography direction.
[0007] Preferably, the antigen-induced release system consists of a urea and a weak acid buffer system with a mass ratio of 1:80-120; the weak acid buffer system is selected from one or more of citrate buffer, acetate buffer, or phosphate buffer.
[0008] Preferably, the weak acid buffer system is a citrate buffer solution with a concentration of 30-50 mM and a pH of 5.0-6.0.
[0009] Preferably, the composite lysis system consists of a nonionic surfactant, an anionic surfactant, and PBS buffer in a mass ratio of 1:0.6-1:30-40; the nonionic surfactant is selected from one or more of Brij-58, Triton X-100, Tween-80, and NP-40; and the anionic surfactant is selected from one or more of sodium deoxycholate, sodium cholate, sodium chenodeoxycholate, and sodium dodecyl sulfate.
[0010] Preferably, the matrix interference blocking system consists of a protein blocker, a metal ion chelating agent, and PBS buffer in a mass ratio of 1:0.3-0.5:300-400; the protein blocker is mouse IgG; and the metal ion chelating agent is selected from one or more of EDTA, citric acid, malic acid, and tartaric acid.
[0011] Preferably, the chromatography promoter is selected from one or more of polyvinylpyrrolidone, polyethylene glycol-6000, and trehalose.
[0012] Preferably, the chromatography promoter is composed of polyvinylpyrrolidone, polyethylene glycol-6000 and trehalose in a mass ratio of 1:0.8-1.2:0.8-1.2.
[0013] Preferably, the method for preparing the antigen pre-enriched particle suspension is as follows: Carboxylated magnetic nanoparticles were dispersed in buffer solution to prepare a particle suspension. EDC and NHS were added to the particle suspension, and the reaction was activated at room temperature in the dark. After activation, magnetic separation was performed, the supernatant was discarded, and the particles were washed to obtain activated magnetic beads. The activated magnetic beads were mixed with anti-Helicobacter pylori antibody, and buffer solution was added to make up to the initial volume of the particle suspension for covalent coupling reaction. After coupling, uncoupled antibody was removed by magnetic separation, and blocking solution was added for blocking treatment. After blocking, blocking solution was removed by magnetic separation, and the particles were washed. Finally, the magnetic beads were resuspended in storage buffer to obtain antigen pre-enriched particle suspension.
[0014] Preferably, the first detection line is coated with 0.5-1.5 μg / cm anti-Helicobacter pylori antibody; the second detection line is coated with 0.3-1.0 μg / cm anti-β-actin antibody; and the control line is coated with 0.5-1.2 μg / cm goat anti-mouse IgG antibody.
[0015] Secondly, this invention provides a method for non-diagnostic applications of a rapid Helicobacter pylori detection kit, employing the following technical solution: A method for using a rapid Helicobacter pylori detection kit for non-diagnostic purposes includes the following steps: S1. Collect fecal samples to be tested; S2. Mix the fecal sample to be tested with the sample processing solution, shake, and obtain the sample processing system. S3. Add the antigen pre-enriched particle suspension to the sample processing system, incubate, remove the supernatant by magnetic separation, resuspend with reconstitution buffer to obtain the test solution; S4. Add the test solution to the sample well of the immunochromatographic test strip and chromatographically analyze at room temperature for 10-15 minutes. S5. Observe the color development of the first test line, the second test line, and the control line, and determine the Helicobacter pylori test result according to the interpretation criteria.
[0016] Preferably, in step S2, the mass-to-volume ratio of the fecal sample to the sample processing liquid is 1:10-20.
[0017] Preferably, in step S3, the volume ratio of the antigen pre-enriched particle suspension to the sample processing system is 1:30-50; the incubation speed is 20-40 rpm, and the incubation time is 8-12 min; the reconstitution buffer is a PBS buffer containing 0.04-0.06% Tween-20 at a concentration of 8-12 mM and a pH of 7.2-7.4; and the volume ratio of the reconstitution buffer to the antigen pre-enriched particle suspension is 4-6:1.
[0018] Preferably, the amount of the test solution added in step S4 is 50-150 μL.
[0019] Preferably, the judgment criteria in step S5 are: If the control line (C line) does not show color, the test is considered invalid regardless of whether the test line shows color. If the control line develops color but the second test line (T2 line) does not, the sample is considered invalid. If the control line (C line) and the second test line (T2 line) show color, but the first test line (T1 line) does not show color, then the result is determined to be negative for Helicobacter pylori. If the control line (C line) shows color, the second test line (T2 line) shows color, and the first test line (T1 line) shows color, then the result is considered positive for Helicobacter pylori.
[0020] In summary, the present invention has the following beneficial effects: (1) Simulating physiological stress to activate endogenous enzyme activity in bacteria and achieve mild and efficient antigen release: This invention uses a weakly acidic buffer system to simulate the gastric microenvironment of Helicobacter pylori. The acidic conditions trigger the acid stress response of the bacteria, which promotes the efficient catalysis of urea decomposition by urease and the in-situ generation of a large amount of ammonia. The ammonia-induced mutation of osmotic pressure inside and outside the bacteria and the sudden change of local pH form an endogenous biochemical shock. It works synergistically with the surfactant in the system to quickly destroy the cell wall and cell membrane of bacteria and efficiently release intracellular antigens. This method abandons extreme physical and chemical treatment methods, protects the spatial conformation and immune epitopes of the antigen to the greatest extent, and allows the released antigen to maintain good natural immune activity, effectively reducing false negative results and greatly improving the sensitivity and accuracy of Helicobacter pylori detection.
[0021] (2) Constructing a composite lysis system to effectively destroy the cell structure of Helicobacter pylori in fecal samples: This invention uses a combination of nonionic and anionic surfactants to form a composite lysis system. The nonionic surfactant can destroy the mucus structure in the fecal sample, and the anionic surfactant can further destroy the bacterial cell membrane structure. The two work together to significantly improve the lysis efficiency of Helicobacter pylori cell structure, thereby further improving the antigen release effect.
[0022] (3) Setting up a matrix interference blocking system to effectively reduce the interference of complex components in fecal samples on the detection reaction: Fecal samples usually contain a large amount of protein, metal ions and other complex substances, which can easily interfere with the immunoassay reaction. This invention establishes a matrix interference blocking system composed of protein blockers and metal ion chelators in the sample processing solution. The protein blockers can reduce non-specific adsorption, and the metal ion chelators can remove metal ion interference, thereby effectively reducing non-specific binding and improving the accuracy and stability of the detection results.
[0023] (4) Setting up an antigen pre-enrichment particle suspension to achieve specific capture and enrichment of low abundance Helicobacter pylori antigen: The present invention uses magnetic nanoparticles with surface-coupled anti-Helicobacter pylori antibody as antigen pre-enrichment particles, which can specifically capture Helicobacter pylori antigen in the sample during the sample processing stage, so that the antigen is enriched before entering the immunochromatographic detection system, thereby improving the detection system's ability to identify low abundance antigen, reducing the false negative rate, and improving detection sensitivity.
[0024] (5) Setting sample validity test lines in the immunochromatographic test strip to achieve effective monitoring of sample quality: This invention sets a first test line, a second test line, and a control line sequentially on a nitrocellulose membrane. The first test line is used to detect Helicobacter pylori antigen, the second test line is coated with anti-β-actin antibody as an internal reference for fecal sample integrity to determine whether the sample contains human intestinal epithelial cell components, and the control line is used to verify whether the chromatographic reaction is normal. By detecting β-actin, invalid samples such as insufficient sample volume, sample degradation, or non-human contamination can be accurately identified, avoiding false negative results caused by invalid samples. At the same time, this invention sets four interpretation criteria: invalid detection, invalid sample, negative, and positive, making the interpretation logic of the test results clear and the operation simple, improving the reliability and operability of the detection process. Detailed Implementation
[0025] The present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.
[0026] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.
[0027] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.
[0028] Among them, the mouse-derived IgG was purchased from Meilun Biotechnology, catalog number: MB2963; Polyvinylpyrrolidone was purchased from MERCK, item number: 81420; Polyethylene glycol-6000 was purchased from Yisheng Biotechnology, product number: 60366ES76; Carboxylated magnetic nanoparticles were purchased from Nanjing JK Biotechnology Co., Ltd., item number: JK-01-001, specification: 50nm; The anti-Helicobacter pylori antibody was purchased from Abcam, catalog number: ab231433; The anti-β-actin antibody was purchased from Wuhan Feien Biotechnology, catalog number: FNab00871; The goat anti-mouse IgG antibody was purchased from Beijing TransGen Biotech, catalog number: HS002-02.
[0029] Example 1 This embodiment provides the composition of a rapid Helicobacter pylori detection kit and its application method for non-diagnostic purposes, as detailed below: A rapid detection kit for Helicobacter pylori includes a sample processing solution, an antigen pre-enriched particle suspension, and an immunochromatographic test strip. The sample processing solution comprises the following raw materials in parts by mass and composition: Ninety samples of antigen-induced release system: composed of urea and citrate buffer (concentration 50 mM, pH 5.0) in a mass ratio of 1:100; Five composite lysis systems were prepared, consisting of Brij-58, sodium deoxycholate, and PBS buffer (50 mM, pH 7.4) in a mass ratio of 1:0.8:35. Three matrix interference blocking systems were prepared: each system consisted of mouse IgG, citric acid, and PBS buffer (50 mM, pH 7.4) in a mass ratio of 1:0.4:350. Chromatography promoter (3 parts): composed of polyvinylpyrrolidone, polyethylene glycol-6000 and trehalose in a mass ratio of 1:1:1.
[0030] The preparation method of antigen pre-enriched particle suspension is as follows: (1) Pretreatment of magnetic beads: Carboxylated magnetic nanoparticles were dispersed in 50mM MES buffer (pH6.0) to prepare a particle suspension of 10mg / mL; (2) Activation reaction: Add EDC and NHS and activate the reaction at room temperature in the dark for 30 min, where the final concentration of EDC is 2 mg / mL and the final concentration of NHS is 2 mg / mL; (3) Washing: After activation, place the magnetic beads on a magnetic rack for magnetic separation, discard the supernatant, and wash twice with 50mM PBS buffer (pH 7.4) to obtain the activated magnetic beads; (4) Antibody conjugation: Mix the two at a ratio of 100 μg anti-Helicobacter pylori antibody per milligram of activated magnetic beads, and then add 50 mM PBS buffer (pH 7.4) to make up to the initial volume of the particle suspension, and covalently conjugate at 20°C for 3 h; (5) Blocking treatment: After conjugation, remove unconjugated antibodies by magnetic separation, add blocking solution (50mM PBS buffer (pH 7.4, containing 1.0wt% BSA) and block at 20℃ for 1h; (6) Washing: After blocking, the blocking solution is removed by magnetic separation, and the solution is washed three times with washing buffer (50mM PBS buffer (pH 7.4, containing 0.05wt% Tween-20); (7) Resuspension and preservation: Finally, the magnetic beads were resuspended in the preservation buffer (50mM PBS (pH7.4, containing 0.1wt% BSA, 0.05wt% Tween-20, 0.05wt% sodium azide and 2wt% sucrose), the magnetic bead concentration was adjusted to 10mg / mL, and stored at 4℃ for later use to obtain the antigen pre-enriched particle suspension.
[0031] The immunochromatographic test strip includes a sample pad, a conjugation pad, a nitrocellulose membrane, and an absorbent pad. A first detection line, a second detection line, and a control line are sequentially arranged along the chromatography direction on the nitrocellulose membrane. The first detection line is coated with 1 μg / cm anti-Helicobacter pylori antibody; the second detection line is coated with 0.5 μg / cm anti-β-actin antibody; and the control line is coated with 1 μg / cm goat anti-mouse IgG antibody.
[0032] A method for using a rapid Helicobacter pylori detection kit for non-diagnostic purposes includes the following steps: S1. Collect fecal samples to be tested; S2. Mix 50 mg of the fecal sample to be tested with 750 μL of sample processing solution, and vortex the mixture for 2 min to obtain the sample processing system. S3. Add 20 μL of antigen pre-enriched particle suspension (10 mg / mL) to the sample processing system, incubate with a reverse mixer at 30 rpm for 10 min, place the reaction tube on a magnetic rack and let it stand for 2 min. After the magnetic beads are completely adsorbed to the tube wall, carefully aspirate the supernatant, add 100 μL of reconstitution buffer (PBS buffer containing 0.05% Tween-20 at a concentration of 10 mM and a pH of 7.4) to resuspend, and obtain the test solution. S4. Add 100 μL of the test solution to the sample well of the immunochromatographic test strip and chromatographically analyze at room temperature for 15 min. S5. Observe the color development of the first test line T1, the second test line T2, and the control line C, and determine the Helicobacter pylori test result according to the interpretation criteria.
[0033] The interpretation criteria are: If the control line (C line) does not show color, the test is considered invalid regardless of whether the test line shows color. If the control line develops color but the second test line (T2 line) does not, the sample is considered invalid. If the control line (C line) and the second test line (T2 line) show color, but the first test line (T1 line) does not show color, then the result is determined to be negative for Helicobacter pylori. If the control line (C line) shows color, the second test line (T2 line) shows color, and the first test line (T1 line) shows color, then the result is considered positive for Helicobacter pylori.
[0034] Example 2 1. Experimental Objective This embodiment aims to verify the synergistic effect of the "antigen-induced release system", "complex lysis system" and "matrix interference blocking system" in the sample processing solution of the present invention on the extraction efficiency, detection sensitivity and specificity of Helicobacter pylori antigen, and at the same time verify the stability protection effect of the sample processing solution on the β-actin internal reference in feces.
[0035] 2. Test Samples ① Low concentration positive samples: Collect 5 fecal samples that were clinically confirmed as positive for Helicobacter pylori by 13C breath test and had a low bacterial load (104 CFU / g), and mix them evenly.
[0036] ② High-concentration interference samples: Five fecal samples from healthy individuals who were clinically confirmed to be free of Helicobacter pylori infection were collected, mixed evenly, and hemoglobin was artificially added to a final concentration of 5 mg / g, and plant fiber (sterilized wheat bran) was artificially added to a final concentration of 5 wt% for testing anti-interference ability.
[0037] 3. Experimental Grouping Except for the sample processing solution formulation, all other reagents and operating procedures were strictly performed according to Example 1. The specific groupings are as follows: ① Experimental group: Same as Example 1.
[0038] ② Control group 1: The antigen-induced release system was removed from the sample processing solution and replaced with an equal volume of 50mM PBS buffer (pH 7.4), while the other components remained unchanged.
[0039] ③ Control group 2: The complex lysis system was removed from the sample processing solution and replaced with an equal volume of 50mM PBS buffer (pH 7.4), while the other components remained unchanged.
[0040] ④ Control group 3: The matrix interference blocking system was removed from the sample processing solution and replaced with an equal volume of 50mM PBS buffer (pH 7.4), while the other components remained unchanged.
[0041] ⑤ Control group 4: Sample processing solution used with commercially available Helicobacter pylori fecal antigen detection kit (Kangwei Century Helicobacter pylori antigen detection kit (colloidal gold method)).
[0042] 4. Testing methods and interpretation standards To comprehensively evaluate the performance of each sample processing solution, each of the five experimental groups described above required independent testing of both "low-concentration positive samples" and "high-concentration interference samples." The detection method was the same as the non-diagnostic application method of the Helicobacter pylori rapid detection kit in Example 1. Each sample type was tested three times in each group, and the average value was taken.
[0043] The interpretation criteria are as follows: ① Color intensity grading: The reflectance density (OD value) of the T1 line, T2 line and C line was measured using a colloidal gold reader.
[0044] "-": OD value < 0.1 (invisible to the naked eye); "+": 0.1 ≤ OD value < 0.3 (faint red line visible to the naked eye); "++": 0.3 ≤ OD value < 0.6 (clear red line to the naked eye); "+++": OD value ≥ 0.6 (deep red line visible to the naked eye).
[0045] ② Background cleanliness: Observe whether there are non-specific colors or trailing phenomena in the background of the test strip.
[0046] ③ Interpretation criteria: Same as in Example 1.
[0047] 5. Experimental Results The detection data and evaluation of low-concentration positive samples in the five groups are shown in Table 1.
[0048] Table 1. Detection results of low-concentration positive samples using different sample treatment solutions. The detection data and evaluation of the high-concentration interference samples in the five groups are shown in Table 2.
[0049] Table 2. Detection results of high-concentration interference samples by different treatment solutions. The results of the comparison between the experimental group and the control group 1 showed that after removing the antigen-induced release system, the colorimetric intensity of the detection of low-concentration positive samples decreased from "+++" to "+", indicating that urea-induced release can significantly improve the extraction efficiency of low-abundance antigens, mainly contributing to the improvement of detection sensitivity.
[0050] The results of the experimental group and the control group 2 showed that after removing the composite lysis system, low-concentration positive samples could not be detected at all (T1 line "-"), indicating that the synergistic lysis of nonionic and anionic surfactants is the core and indispensable link in antigen release.
[0051] The results of the comparison between the experimental group and the control group 3 showed that after removing the matrix interference blocking system, obvious false positives (T1 line "+") and background color trailing appeared when processing high concentration interference samples. This indicates that the dual-mechanism blocking of protein blocking agents and metal ion chelating agents is the key to maintaining detection specificity and can effectively inhibit non-specific adsorption caused by interferences such as hemoglobin and divalent cations.
[0052] In summary, the experimental group performed best across all indicators, detecting strong positives in low-concentration samples and accurately detecting negatives in high-concentration interference samples with a clean background. It also stably detected β-actin internal reference to confirm sample validity. Overall, its performance was superior to the control group lacking any system and commercially available products.
[0053] Example 3 1. Experimental Objective This embodiment aims to verify the enrichment effect of the antigen pre-enriched particle suspension on Helicobacter pylori antigen in this invention, and to prove that this step can improve the detection sensitivity of low abundance samples.
[0054] 2. Test Samples Preparation of serial concentrations of Helicobacter pylori-positive samples: Fecal samples clinically confirmed as Helicobacter pylori-positive by 13C breath test were collected and serially diluted with fecal samples from healthy individuals confirmed to be free of Helicobacter pylori infection to prepare test samples at the following three concentration levels: High-concentration positive sample: 10 6 CFU / g; Medium-concentration positive samples: 10 5 CFU / g; Low concentration positive sample: 10 4 CFU / g 3. Experimental Grouping Except for the use of antigen pre-enriched particle suspension, all other reagents and procedures were strictly performed according to Example 1. Specific groupings are as follows: ① Experimental groups 1-3: Same as Example 1 (high, medium and low concentration positive samples were detected respectively).
[0055] ② Control group 1-3: After the sample processing solution is completed, without antigen pre-enrichment particle enrichment, the supernatant is directly taken as the test solution for detection (high, medium and low concentration positive samples are detected respectively).
[0056] ③ Positive control group: Known strong positive samples were detected using the complete method described in Example 1, and used as a reference for colorimetric intensity.
[0057] ④ Negative control group: Negative stool samples from healthy individuals were tested using the complete method described in Example 1 as a background reference.
[0058] 4. Testing methods and interpretation standards Each of the four experimental groups described above requires independent testing of "high concentration," "medium concentration," and "low concentration" positive samples. The detection method is the same as the non-diagnostic application method of the Helicobacter pylori rapid test kit in Example 1. Each sample concentration is tested three times in each group, and the average value is taken.
[0059] The interpretation criteria are as follows: ① Color intensity grading: Same as in Example 2.
[0060] ②Detection time: Record the time from the addition of the test solution to the appearance of a clear color on the T1 line.
[0061] ③ Interpretation criteria: Same as in Example 1.
[0062] 5. Experimental Results The detection data of each group for positive samples of different concentrations are shown in Table 3.
[0063] Table 3. Detection results of antigen pre-enrichment steps on positive samples of different concentrations. As shown in Table 3, the antigen pre-enrichment step described in this invention significantly improves detection sensitivity. For high-concentration positive samples, both the experimental and control groups were able to detect positive results, but the experimental group had a shorter detection time. For medium-concentration positive samples, the experimental group showed higher color intensity, indicating that the enrichment step effectively improved signal intensity. For low-concentration positive samples, the experimental group was still able to clearly detect positive results, while the control group could not detect them and was interpreted as negative. The results of the positive and negative control groups were normal, verifying the effectiveness of the detection system. The above results indicate that by specifically capturing and enriching Helicobacter pylori antigen in samples using antigen pre-enrichment particles, the detection sensitivity can be increased by approximately 10 times, which is particularly suitable for screening individuals with low viral loads and effectively reduces the false negative rate. Simultaneously, the magnetic separation and washing process in the enrichment step can remove some impurities, resulting in a cleaner detection background and clearer color development. Meanwhile, the T2 line (β-actin internal reference) of all test groups showed stable color development, proving that the magnetic separation and incubation operations of antigen pre-enrichment do not destroy the β-actin internal reference in feces, and the sample validity monitoring system can play a stable role throughout the entire testing process.
[0064] Example 4 1. Experimental Objective This embodiment aims to verify whether the second detection line (T2 line, anti-β-actin antibody) set in the immunochromatographic test strip can effectively identify invalid samples caused by insufficient sampling, avoid false negative results, and prove the practical value of the "dual-line quality control + four-class interpretation" system of the present invention.
[0065] 2. Test Samples ① Normal sampling positive group: Collect 10 stool samples that were clinically confirmed as positive for Helicobacter pylori by 13C breath test. Each sample was collected at the routine sampling amount (50mg) and mixed evenly.
[0066] ②Normal sampling negative group: Collect 10 fecal samples from healthy individuals who were clinically confirmed to be free of Helicobacter pylori infection. Each sample was collected at the standard sampling amount (50mg) and mixed thoroughly.
[0067] ③ Micro-sampling positive group: Take 10 positive samples from the same source as the normal sampling positive group, and use only the tip of the sampling stick to take a micro amount (5mg, 1 / 10 of the normal sampling amount) to simulate the situation of insufficient user sampling, and mix them evenly.
[0068] 3. Experimental Grouping Except for the different test strip structure, all other reagents and operating procedures were strictly performed according to Example 1. The specific groupings are as follows: ① Experimental Groups 1-3: Complete test strip cards containing the first detection line (T1 line), the second detection line (T2 line), and the control line (C line) prepared in Example 1.
[0069] ② Control groups 1-3: Test strips prepared in the same batch but containing only the first detection line (T1 line) and the quality control line (C line), without the second detection line T2, to simulate commercially available traditional products.
[0070] 4. Testing methods and interpretation standards Each of the two experimental groups described above requires independent testing of samples from the "normal sampling positive group," "normal sampling negative group," and "trace sampling positive group." The detection method is the same as the non-diagnostic application method of the Helicobacter pylori rapid test kit in Example 1. Each sample type is tested three times in each group, and the average value is taken.
[0071] The interpretation criteria are as follows: ① Color development record: Record the color development of the first detection line (T1 line), the second detection line (T2 line), and the quality control line (C line) respectively ("+" indicates color development, "-" indicates no color development).
[0072] ② Interpretation criteria: Same as in Example 1.
[0073] 5. Experimental Results The detection data and evaluations for different sample types in each group are shown in Table 4.
[0074] Table 4. Detection results of dual detection lines for different sample types. As can be seen from the experimental results in Table 4, the second detection line (T2 line) set in this invention plays an important role in monitoring sample validity. For the positive and negative groups with normal sampling, the interpretation results of the experimental group and the control group were completely consistent, both being 10 / 10 correct, indicating that the presence of the T2 line does not affect normal detection.
[0075] For the positive group with micro-sampling, 10 samples (10 / 10) in the experimental group were accurately interpreted as "invalid samples" because the T2 line did not develop, prompting the user to resample, effectively avoiding false negative results caused by insufficient sampling. In contrast, the control group, lacking a T2 line, had all 10 micro-sampling samples misjudged as "negative," resulting in a serious false negative. These results demonstrate that this invention, by setting a second detection line to detect β-actin, can effectively identify invalid samples caused by insufficient sampling, significantly improving the reliability of the test results, especially suitable for home self-testing scenarios where user operation is not standardized.
[0076] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.
Claims
1. A rapid detection kit for Helicobacter pylori, characterized in that, Includes sample processing solution, antigen pre-enriched particle suspension, and immunochromatographic test strips; The sample processing solution comprises the following raw materials in parts by weight: 80-95 parts of antigen-induced release system, 2-8 parts of complex lysis system, 1-5 parts of matrix interference blocking system, and 2-3 parts of chromatography promoter; The antigen pre-enriched particles in the antigen pre-enriched particle suspension are magnetic nanoparticles with anti-Helicobacter pylori antibodies coupled to their surface. The immunochromatographic test strip includes a sample pad, a conjugation pad, a nitrocellulose membrane, and an absorbent pad. The nitrocellulose membrane has a first detection line, a second detection line, and a control line arranged sequentially along the chromatography direction.
2. The rapid detection kit for Helicobacter pylori according to claim 1, characterized in that, The antigen-induced release system consists of a urea and a weak acid buffer system with a mass ratio of 1:80-120; the weak acid buffer system is selected from one or more of citrate buffer, acetate buffer, or phosphate buffer.
3. The rapid detection kit for Helicobacter pylori according to claim 1, characterized in that, The composite lysis system consists of a nonionic surfactant, an anionic surfactant, and PBS buffer in a mass ratio of 1:0.6-1:30-40; the nonionic surfactant is selected from one or more of Brij-58, Triton X-100, Tween-80, and NP-40; the anionic surfactant is selected from one or more of sodium deoxycholate, sodium cholate, sodium chenodeoxycholate, and sodium dodecyl sulfate.
4. The rapid detection kit for Helicobacter pylori according to claim 1, characterized in that, The matrix interference blocking system consists of a protein blocker, a metal ion chelating agent, and PBS buffer in a mass ratio of 1:0.3-0.5:300-400; the protein blocker is mouse IgG; and the metal ion chelating agent is selected from one or more of EDTA, citric acid, malic acid, and tartaric acid.
5. The rapid detection kit for Helicobacter pylori according to claim 1, characterized in that, The chromatography promoter is selected from one or more of polyvinylpyrrolidone, polyethylene glycol-6000, and trehalose.
6. The rapid detection kit for Helicobacter pylori according to claim 1, characterized in that, The method for preparing the antigen pre-enriched particle suspension is as follows: carboxylated magnetic nanoparticles are dispersed in buffer solution to prepare a particle suspension; EDC and NHS are added to the particle suspension, and the activation reaction is carried out at room temperature in the dark; after activation, magnetic separation is performed and the supernatant is discarded, and the mixture is washed to obtain activated magnetic beads; the activated magnetic beads are mixed with anti-Helicobacter pylori antibody, and then buffer solution is added to make up to the initial volume of the particle suspension for covalent coupling reaction. After conjugation, unconjugated antibodies are removed by magnetic separation, and blocking buffer is added for blocking. After blocking, the blocking buffer is removed by magnetic separation and the mixture is washed. Finally, the magnetic beads were resuspended in the storage buffer to obtain an antigen pre-enriched particle suspension.
7. The rapid detection kit for Helicobacter pylori according to claim 1, characterized in that, The first test line is coated with 0.5-1.5 μg / cm of anti-Helicobacter pylori antibody; the second test line is coated with 0.3-1.0 μg / cm of anti-β-actin antibody; and the control line is coated with 0.5-1.2 μg / cm of goat anti-mouse IgG antibody.
8. A method of using the Helicobacter pylori rapid detection kit according to any one of claims 1-7 for non-diagnostic purposes, characterized in that, Includes the following steps: S1. Collect fecal samples to be tested; S2. Mix the fecal sample to be tested with the sample processing solution, shake, and obtain the sample processing system. S3. Add the antigen pre-enriched particle suspension to the sample processing system, incubate, remove the supernatant by magnetic separation, resuspend with reconstitution buffer to obtain the test solution; S4. Add the test solution to the sample well of the immunochromatographic test strip and chromatographically analyze at room temperature for 10-15 minutes. S5. Observe the color development of the first test line, the second test line, and the control line, and determine the Helicobacter pylori test result according to the interpretation criteria.
9. The method of using the Helicobacter pylori rapid detection kit according to claim 8 for non-diagnostic purposes, characterized in that, In step S2, the mass-to-volume ratio of the fecal sample to be tested to the sample processing liquid is 1:10-20.
10. The method of using the Helicobacter pylori rapid detection kit according to claim 8 for non-diagnostic purposes, characterized in that, The judgment criteria in step S5 are as follows: If the control line does not show color, the test is considered invalid regardless of whether the test line shows color. If the control line shows color but the second test line does not, the sample is considered invalid. If the control line shows color and the second test line shows color, but the first test line does not show color, then the result is determined to be negative for Helicobacter pylori. If the control line, the second test line, and the first test line all show color, then the result is considered positive for Helicobacter pylori.