Helicobacter pylori detection kit based on urease method and preparation method
By constructing a high buffer capacity system using a specific concentration of phosphate buffer and microporous modified trehalose, and setting up negative control wells, the false positive problem in the urease method for Helicobacter pylori detection was solved, achieving high accuracy and high sensitivity in detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHONGQING XINSAIYA BIOTECHNOLOGY CO LTD
- Filing Date
- 2025-12-26
- Publication Date
- 2026-05-15
AI Technical Summary
In existing urease-based Helicobacter pylori testing, dental plaque samples suffer from high false positive rates and poor accuracy due to the complex oral environment and lack of control systems, making it difficult to meet the needs of precision medicine.
A high buffer capacity system was constructed using phosphate buffer solution of a specific concentration and microporous modified trehalose. A negative control well was set up, and the sample was spread evenly by guiding the sample through the sample loading groove. The three-dimensional structure of microporous trehalose adsorbed urea molecules and the capillary action achieved rapid reaction.
It significantly reduces the false positive rate, improves detection accuracy and sensitivity, with a positive concordance rate of 96.88%-100% and a negative concordance rate of 95.52%-97.01%, and provides rapid response to weakly positive samples.
Smart Images
Figure CN122038533A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pathogenic microorganism detection technology, and in particular to a Helicobacter pylori detection kit and its preparation method based on the urease method. Background Technology
[0002] Helicobacter pylori (Hp) is an S-shaped or L-shaped Gram-negative bacterium that colonizes the gastric mucosa pits and adjacent surface epithelium via flagellar motility. Hp infection is closely associated with the development of various upper gastrointestinal diseases, including gastritis, peptic ulcers, and gastric cancer. The infection rate exceeds 95% in patients with chronic gastritis, and it is classified as a Group 1 carcinogen by the World Health Organization. In my country, approximately 50-60% of the population is infected with Hp. Because persistent Hp infection can lead to precancerous lesions such as intestinal metaplasia and dysplasia, rapid and convenient detection of Hp is of paramount importance for clinical diagnosis and subsequent treatment.
[0003] Currently, various methods have been developed for the clinical diagnosis of Helicobacter pylori infection, mainly including microbiological methods, serological methods, gene diagnosis, and urease-dependent techniques. Among these, the rapid urease test is based on the principle that *H. pylori* produces highly active urease, which breaks down urea in gastric acid to produce ammonia (NH4) and carbon dioxide. The ammonia increases the pH of the environment, causing the phenol red indicator in the reagent to change from yellow to red. Observing this color change indicates the presence of infection. Because this method is simple to perform and has a short observation time, it has important auxiliary diagnostic value for clinical diagnosis of *H. pylori* infection.
[0004] Existing urease-based detection methods primarily use gastric mucosa and dental plaque as samples. While dental plaque samples offer advantages such as convenience, speed, and on-demand testing, current technologies still have significant drawbacks. First, the oral environment is complex and highly susceptible to dietary influences, resulting in dental plaque samples with diverse compositions. Second, commercially available urease-based dental plaque detection kits almost never include control systems, making it difficult to eliminate interference from non-specific factors. This leads to false positives in practical applications, resulting in low overall accuracy and failing to meet the demands of precision medicine. Summary of the Invention
[0005] In view of this, the purpose of this invention is to provide a Helicobacter pylori detection kit and preparation method based on the urease method, which can address the problems of high false positive rate, poor accuracy and slow reagent response speed in the detection of Helicobacter pylori using dental plaque samples in the prior art, due to the complex oral environment (such as food residue, non-specific bacterial interference) and lack of an effective control system.
[0006] The present invention solves the above-mentioned technical problems through the following technical means:
[0007] In a first aspect, the present invention provides a Helicobacter pylori detection kit based on the urease method, comprising a detection kit, a test strip, and a dental plaque sampling tag. During the preparation process, the test strip is soaked in a reaction solution and a buffer solution. The buffer solution has a pH of 6.5-7.0 and comprises potassium dihydrogen phosphate and dipotassium hydrogen phosphate, wherein the final concentration of potassium dihydrogen phosphate is 14-30 g / L and the final concentration of dipotassium hydrogen phosphate is 1.8-3.6 g / L.
[0008] Preferably, the reaction solution includes phenol red and urea, wherein the final concentration of urea is 45-50 g / L and the final concentration of phenol red is 0.5-0.7 g / L.
[0009] Preferably, the reaction solution further includes a stabilizer and a penetrant, wherein the stabilizer is microporous trehalose and the penetrant is Triton X-100 or Tween-20; the final concentration of microporous trehalose is 8-12 g / L and the final concentration of penetrant is 0.8-1.2 g / L.
[0010] Preferably, the preparation method of the microporous trehalose includes the following steps: preparing a trehalose aqueous solution with a final concentration of 100-200 g / L, adding a pore-forming template agent to the trehalose aqueous solution, stirring to dissolve, and freeze-drying the mixed solution to obtain a porous block; placing the lightweight porous trehalose block under a ventilated condition at 40-50℃ for descaling treatment for 1-2 hours, and then pulverizing and sieving to obtain the microporous trehalose.
[0011] Preferably, the pore-forming template agent is sodium bicarbonate with a final concentration of 2-8 g / L or ammonium bicarbonate with a final concentration of 1-5 g / L, and the sieving is through a 50-100 mesh sieve.
[0012] Preferably, the surface of the test kit is provided with two shallow circular holes and a sample application groove. The two shallow circular holes are a test hole and a negative control hole, respectively, and the sample application groove is used to guide the uniform application of the sample.
[0013] Secondly, the present invention provides a method for preparing a Helicobacter pylori detection kit based on the urease method, which includes the following steps:
[0014] S1. Weigh potassium dihydrogen phosphate and dipotassium hydrogen phosphate, add purified water, and prepare phosphate buffer solution;
[0015] S2. Weigh out urea, phenol red, stabilizer and penetrant, add to the phosphate buffer solution to prepare a mixture, and heat and stir.
[0016] S3. The test strip is immersed in the mixture for wetting, and then dried to obtain the test strip.
[0017] S4. Cut the test strip for later use. Assemble the cut test strip into the test sleeve and attach a transparent film to the plastic card surface where the sample application hole is located.
[0018] Preferably, in step S1, the phosphate buffer solution needs to be stirred until there is no precipitate.
[0019] Preferably, in step S2, the heating and stirring temperature is 55-65℃, and the stirring time is 10-15 minutes.
[0020] Preferably, in step S3, the soaking time of the test paper in the mixed solution is 15-20 minutes, and the drying conditions are: drying at 35-50°C for 50-120 minutes.
[0021] The beneficial effects of this invention are:
[0022] (1) This invention constructs a high buffer capacity system by screening specific phosphate buffer concentration ratios. This system can effectively resist pH fluctuations caused by ammonia produced by non-specific bacteria in the oral cavity, and establishes a "chemical threshold" to shield against false positives. Experimental data show that the positive concordance rate of the test strip of this invention can reach 96.88%-100%, and the negative concordance rate can reach 95.52%-97.01%.
[0023] (2) The kit is equipped with a negative control well, which can directly compare and eliminate the interference of the oral background environment (such as alkaline food residue); the specially designed sample loading groove guides the user to evenly apply the irregular dental plaque sample to the reaction center, avoiding uneven reaction caused by sample accumulation, and further improving the convenience of operation and the reliability of results.
[0024] (3) This invention introduces “microporous modified trehalose” prepared by the bicarbonate template method. Its unique three-dimensional microporous structure has an extremely high specific surface area. On the one hand, it can adsorb and stabilize urea molecules in a dry state to prevent them from oxidizing and becoming ineffective. On the other hand, during detection, it can rapidly absorb moisture from dental plaque using capillary action to achieve “instantaneous resolution” of the reaction system, which significantly shortens the reaction lag time and improves the detection sensitivity for weak positive samples. Attached Figure Description
[0025] Figure 1 This is a schematic diagram of the structure of the present invention;
[0026] Figure 2 This is an assembly diagram of the Hp test strip of the present invention. Detailed Implementation
[0027] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0028] Example 1
[0029] The preparation method of microporous trehalose includes the following steps:
[0030] Weigh 10g of trehalose and add it to 100mL of deionized water. After dissolving it by magnetic stirring, a trehalose solution is formed. Add 0.1g of NH4HCO3 to the trehalose solution and continue stirring for 10min to fully dissolve it, obtaining a trehalose precursor solution containing a pore-forming template. Pre-freeze the precursor solution at -40℃ for 2h, and then freeze-dry it in a vacuum freeze dryer for 24h to obtain a lightweight porous trehalose block. Place the lightweight porous trehalose block under ventilated conditions at 40℃ for 2 hours to remove impurities. After pulverizing and passing it through a 50-sieve, 14.95g of microporous trehalose is obtained.
[0031] A method for preparing a Helicobacter pylori detection kit based on the urease method includes the following steps:
[0032] S1. Weigh 14.38g of potassium dihydrogen phosphate and 1.85g of dipotassium hydrogen phosphate, add 1000mL of purified water, and prepare a phosphate buffer solution;
[0033] S2. Weigh 40.03g urea, 0.58g phenol red, 8.12g microporous trehalose and 0.83g Triton X-100, add 1000mL phosphate buffer to prepare a mixture, heat and stir;
[0034] S3. The test strip is immersed in the mixture for wetting, and then dried to obtain the test strip.
[0035] S4. Cut the test strip to 1.3cm×1.3cm and set aside. Assemble the cut test strip into the test sleeve and attach a transparent film to the plastic card surface where the sample application hole is located.
[0036] Example 2
[0037] The preparation method of microporous trehalose includes the following steps:
[0038] Weigh 15g of trehalose and add it to 100mL of deionized water. After dissolving it by magnetic stirring, a trehalose solution is formed. Add 0.3g of NH4HCO3 to the trehalose solution and continue stirring for 10min to fully dissolve it, obtaining a trehalose precursor solution containing a pore-forming template. Pre-freeze the precursor solution at -40℃ for 2h, and then freeze-dry it in a vacuum freeze dryer for 24h to obtain a lightweight porous trehalose block. Place the lightweight porous trehalose block under ventilated conditions at 45℃ for 1.5h for descaling, and then pulverize it through a 75-sieve to obtain 14.37g of microporous trehalose.
[0039] A method for preparing a Helicobacter pylori detection kit based on the urease method includes the following steps:
[0040] S1. Weigh 22g of potassium dihydrogen phosphate and 2.7g of dipotassium hydrogen phosphate, add 1000mL of purified water, and prepare a phosphate buffer solution.
[0041] S2. Weigh 47.5g urea, 0.6g phenol red, 10g microporous trehalose and 1g Triton X-100, add 1000mL phosphate buffer to prepare a mixture, heat and stir.
[0042] S3. The test strip is immersed in the mixture for wetting, and then dried to obtain the test strip.
[0043] S4. Cut the test strip to 1.3cm×1.3cm and set aside. Assemble the cut test strip into the test sleeve and attach a transparent film to the plastic card surface where the sample application hole is located.
[0044] Example 3
[0045] The preparation method of microporous trehalose includes the following steps:
[0046] Weigh 20g of trehalose and add it to 100mL of deionized water. After dissolving it with magnetic stirring, a trehalose solution is formed. Add 0.5g of NH4HCO3 to the trehalose solution and continue stirring for 10min to fully dissolve it, obtaining a trehalose precursor solution containing a pore-forming template. Pre-freeze the precursor solution at -40℃ for 2h, and then freeze-dry it in a vacuum freeze dryer for 24h to obtain a lightweight porous trehalose block. Place the lightweight porous trehalose block under 50℃ ventilation for 1 hour to remove impurities. After crushing and passing it through a 100-sieve, 13.78g of microporous trehalose is obtained.
[0047] A method for preparing a Helicobacter pylori detection kit based on the urease method includes the following steps:
[0048] S1. Weigh 29.5g of potassium dihydrogen phosphate and 3.55g of dipotassium hydrogen phosphate, add 1000mL of purified water, and prepare a phosphate buffer solution;
[0049] S2. Weigh 49.23g urea, 0.65g phenol red, 11.76g microporous trehalose and 1.18g Triton X-100, add 1000mL phosphate buffer to prepare a mixture, heat and stir;
[0050] S3. The test strip is immersed in the mixture for wetting, and then dried to obtain the test strip.
[0051] S4. Cut the test strip to 1.3cm×1.3cm and set aside. Assemble the cut test strip into the test sleeve and attach a transparent film to the plastic card surface where the sample application hole is located.
[0052] Example 4
[0053] The difference from Example 2 is that the pore-forming template agent is 5 g / L sodium bicarbonate, and the penetrant is Tween-20.
[0054] Comparative Example 1
[0055] The difference from Example 1 is that the amount of urea added is 27.55g and the amount of phenol red added is 0.1g.
[0056] Comparative Example 2
[0057] The difference from Example 1 is that the amount of urea added is 70.23g and the amount of phenol red added is 2.64g.
[0058] Comparative Example 3
[0059] The difference from Example 3 is that the amount of potassium dihydrogen phosphate added is 5.02g and the amount of phenol red added is 0.65g.
[0060] Comparative Example 4
[0061] The difference from Example 3 is that the amount of potassium dihydrogen phosphate added is 4.55g and the amount of phenol red added is 35.8g.
[0062] Comparative Example 5
[0063] The difference from Example 2 is that microporous trehalose is replaced with ordinary trehalose.
[0064] Comparative Example 6
[0065] The difference from Example 2 is that the trehalose solution was only freeze-dried.
[0066] Hp detection methods
[0067] When conducting Hp infection testing, C14 breath test results were used as a standard control method to determine whether or not Hp infection was present. Dental plaque samples from 32 individuals who tested positive for C14 breath test results and 67 individuals who tested negative for C14 breath test results were selected as research subjects. The test strips of Examples 1 and 2 and Comparative Examples 1, 2, 3, and 4 were used to test 99 dental plaque samples to determine whether Hp infection was present.
[0068] The test strip detection method is as follows:
[0069] 1. Gently peel off the transparent film from the Hp test kit to the yellow circular reaction area;
[0070] 2. Take a tartar sampling stick and collect 2-3 visible tartar samples from inside your mouth. Apply the samples to the center of the circular reaction area.
[0071] 3. Close the transparent film and observe the color change within 3 minutes.
[0072] Note: Do not eat or drink acidic or alkaline water for two hours before sampling.
[0073] Result interpretation: If the test strip changes from yellow to red, it indicates Hp positivity; if it does not change color, it indicates Hp positivity.
[0074] The test results are shown in Tables 1 and 2:
[0075] Table 1
[0076]
[0077] Note: "+" indicates a positive Hp test, and "-" indicates a negative Hp test.
[0078] Table 2
[0079]
[0080] As shown in Tables 1 and 2, Examples 1-4 using the technical solution of this invention all exhibited excellent detection performance, with consistency with the C14 breath test exceeding 95%. This indicates that the buffer concentration range determined by this invention, combined with the micropore trehalose technology, can achieve a balance between high sensitivity and high specificity. Example 4 demonstrates that changing the pore template agent (sodium bicarbonate) and adding the permeabilizer (Tween-20) also maintains excellent detection results, proving the universality of the technical solution.
[0081] 1. The decisive effect of buffer concentration on "false positives" (Comparative Example 3 vs. Comparative Example 3):
[0082] While the positive concordance rate of Comparative Example 3 (low concentration of potassium dihydrogen phosphate 5.02 g / L) was 100%, the negative concordance rate was extremely low, at only 25.37%. This means that approximately 50 out of 67 negative samples were falsely identified as positive. This is because the buffer concentration was too low, resulting in insufficient buffer capacity and an inability to shield against trace amounts of ammonia produced by oral bacteria, leading to an extremely high false positive rate.
[0083] In contrast, Example 3 used the high-concentration buffer solution required by this invention, and the negative compliance rate was improved to 97.01%. This strongly demonstrates that the specific phosphate concentration range of this invention constructs an effective "chemical threshold" that successfully filters out non-specific interference.
[0084] Comparative Example 4 shows that when the buffer solution ratio is unbalanced (e.g., the component deviation is too large), although there are no false positives (100% negative concordance rate), the positive reaction is severely suppressed (positive concordance rate is only 62.50%), resulting in a large number of missed detections.
[0085] 2. The effect of trehalose microporous structure on "sensitivity" (Comparative Example 2 and Comparative Examples 5 and 6):
[0086] The positive concordance rate of Comparative Example 5 (ordinary trehalose) was the lowest, at only 78.13%. Ordinary trehalose dissolves slowly and has poor permeability, which means that weakly positive samples cannot develop color within the specified time, resulting in a large number of false negatives (missed detections).
[0087] The positive concordance rate of Comparative Example 6 (lyophilized trehalose alone) increased to 90.63%, indicating that the loose structure brought about by the lyophilization process is helpful to some extent, but some weakly positive samples were still missed.
[0088] Examples 2-3 (microporous modified trehalose) improved the positive concordance rate to 96.88%-100%. This demonstrates that by adding a pore-forming template agent (such as ammonium bicarbonate / sodium bicarbonate) to construct a connected microporous structure inside trehalose, the reaction system can achieve "instantaneous resolubilization," thereby keenly capturing weak positive samples with low bacterial load and significantly improving detection sensitivity.
[0089] 3. Effect of substrate concentration (Comparative Example 1 and Comparative Examples 1 and 2):
[0090] The detection accuracy of Comparative Example 1 (low urea / phenol red) and Comparative Example 2 (high urea / phenol red) was significantly lower than that of Example 1. This indicates that the concentration of each component in the reaction solution must be controlled within the specific ratio range defined in this invention. Too low a concentration will result in unclear color development, while too high a concentration may cause substrate inhibition or background color interference, both of which will reduce the reliability of the detection results.
[0091] In summary, this invention, through the synergistic effect of "specific high-concentration buffer solution" and "microporous modified trehalose," effectively solves the problem of interference from oral bacteria (eliminating false positives) while ensuring a rapid response to weakly positive samples (eliminating false negatives), thus solving the problem of existing technologies struggling to balance accuracy and sensitivity.
[0092] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.
Claims
1. A Helicobacter pylori detection kit based on the urease method, characterized in that, The test kit includes a test strip and a dental plaque sampling tag. The test strip is soaked in a reaction solution and a buffer solution during the preparation process. The buffer solution has a pH of 6.5-7.0 and includes potassium dihydrogen phosphate and dipotassium hydrogen phosphate. The final concentration of potassium dihydrogen phosphate is 14-30 g / L and the final concentration of dipotassium hydrogen phosphate is 1.8-3.6 g / L.
2. The reagent kit according to claim 1, characterized in that, The reaction solution includes phenol red and urea, wherein the final concentration of urea is 45-50 g / L and the final concentration of phenol red is 0.5-0.7 g / L.
3. The reagent kit according to claim 2, characterized in that, The reaction solution also includes a stabilizer and a penetrant. The stabilizer is microporous trehalose, and the penetrant is Triton X-100 or Tween-20. The final concentration of microporous trehalose is 8-12 g / L, and the final concentration of the penetrant is 0.8-1.2 g / L.
4. The reagent kit according to claim 3, characterized in that, The preparation method of the microporous trehalose includes the following steps: preparing a trehalose aqueous solution with a final concentration of 100-200 g / L, adding a pore-forming template agent to the trehalose aqueous solution, stirring to dissolve, and freeze-drying the mixed solution to obtain a porous block; placing the lightweight porous trehalose block under a ventilated condition at 40-50℃ for descaling treatment for 1-2 hours, and then pulverizing and sieving to obtain the microporous trehalose.
5. The reagent kit according to claim 4, characterized in that, The pore-forming template agent is sodium bicarbonate with a final concentration of 2-8 g / L or ammonium bicarbonate with a final concentration of 1-5 g / L, and the sieving is passing through a 50-100 mesh sieve.
6. The reagent kit according to claim 1, characterized in that, The test kit has two shallow circular holes and a sample application groove on its surface. The two shallow circular holes are the test hole and the negative control hole, respectively, and the sample application groove is used to guide the uniform application of the sample.
7. A method for preparing a Helicobacter pylori detection kit based on the urease method, used to prepare the kit according to any one of claims 1-6, characterized in that, Includes the following steps: S1. Weigh potassium dihydrogen phosphate and dipotassium hydrogen phosphate, add purified water, and prepare phosphate buffer solution; S2. Weigh out urea, phenol red, stabilizer and penetrant, add to the phosphate buffer solution to prepare a mixture, and heat and stir. S3. Immerse the test paper in the mixture for wetting, and then dry it to obtain the test paper; S4. Cut the test strip for later use. Assemble the cut test strip into the test sleeve and attach a transparent film to the plastic card surface where the sample application hole is located.
8. The preparation method according to claim 7, characterized in that, In step S1, the phosphate buffer solution needs to be stirred until there is no precipitate.
9. The preparation method according to claim 7, characterized in that, In step S2, the heating and stirring temperature is 55-65℃, and the stirring time is 10-15 minutes.
10. The preparation method according to claim 7, characterized in that, In step S3, the soaking time of the test paper in the mixed solution is 15-20 minutes, and the drying conditions are: drying at 35-50℃ for 50-120 minutes.