A Helicobacter pylori detection reagent based on oral sampling and its use method

Abstract

The present invention relates to the technical field of biological detection reagents, and in particular to a Helicobacter pylori detection reagent based on oral sampling and a method for using the same; the Helicobacter pylori detection reagent contains components such as an antibacterial agent, an enzyme activator, a nutrient, etc., has a pH value of 7.6 and is sealed and stored in the form of a dry powder, can effectively detect Helicobacter pylori, and has better specificity, sensitivity and stability than other similar products.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection reagents, and more specifically, to a Helicobacter pylori detection reagent based on oral sampling and a use method thereof. Background Art

[0002] Early screening and diagnosis of Helicobacter pylori (HP) infection is the prerequisite for standardized treatment of H. pylori and is of great significance for the occurrence of gastric inflammation and gastric cancer. According to whether the detection method requires endoscopic examination, the detection methods of H. pylori can be divided into invasive and non-invasive detection. Invasive detection includes direct smear staining of gastric mucosal specimens, histological staining, rapid urease test, bacterial culture, HP-specific gene detection, etc., but invasive detection is a detection method that damages the body and often requires endoscopic examination as an aid. Non-invasive detection includes breath test, fecal antigen test, serological test, etc., which has the characteristics of not causing damage to the stomach and has high acceptance. Although invasive detection has high sensitivity, it requires high operating experience of doctors, high equipment and equipment costs, and patients need to endure the pain of inserting the scope, low patient compliance, and easy cross-infection.

[0003] At present, how to quickly, accurately, efficiently and conveniently screen Helicobacter pylori HP is still a problem that industry technicians need to study and solve. Moreover, the HP tests currently on the market are more likely to have false negatives or false positives when testing oral samples compared to other samples, because the oral environment is relatively more complex and is greatly affected by food. Summary of the invention

[0004] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a Helicobacter pylori detection reagent based on oral sampling; the detection reagent provided by the present invention can effectively detect Helicobacter pylori and has better specificity, sensitivity and stability than other similar products.

[0005] To achieve the above object, the present invention is implemented through the following technical solutions:

[0006] A Helicobacter pylori detection reagent based on oral sampling, the preparation method of which comprises the following steps:

[0007] S1: Evenly mix the buffer solution, the antibacterial agent, the enzyme activator and the nutrient agent to obtain a first solution;

[0008] S2: adding the detection mother solution to the first solution and mixing them evenly to obtain a second solution;

[0009] S3: transferring the second solution into a reagent tube and drying it into a powder to obtain a test powder;

[0010] S4: Heat-seal the detection powder and the desiccant in a light-proof aluminum foil bag to obtain the Helicobacter pylori detection reagent.

[0011] Preferably, the pH value of the second solution is 7.6.

[0012] Preferably, the buffer comprises the following components in mass percentage: 0.5-1.5% sodium chloride, 0.02-0.05% potassium chloride, 0.5-1.5% potassium dihydrogen phosphate, 0.5-1.5% disodium hydrogen phosphate, 0.2-0.6% sodium hydroxide, and the balance pure water.

[0013] Preferably, the antibacterial agent comprises the following components in percentage by mass: 10-12% vancomycin, 8-10% trimethylolpropane, 4-6% amphotericin B, and the balance pure water.

[0014] Preferably, the enzyme activator is magnesium sulfate, and its content is 2-10 mM.

[0015] Preferably, the detection mother solution comprises the following components in percentage by mass: 10-12% urea, 0.01-0.02% phenol red, and the balance pure water.

[0016] Preferably, in step S3, the second solution is dried to powder at 55-60°C.

[0017] The design ideas of the Helicobacter pylori detection reagent in the present invention are as follows:

[0018] In order to make urease obtain the highest reaction activity, the system pH value of the detection reagent is set to 7.6, because the optimal reaction pH value of urease is 7.6; in order to maintain the pH value of the system at 7.6, the present invention adopts a buffer composed of a phosphate buffer solution and a sodium hydroxide solution; in order to make the detection reagent highly specific, the present invention uses vancomycin, trimethylolpropane (TMP), amphotericin B and the like as antibacterial agents to prevent miscellaneous bacteria from participating in the reaction; in order to make the detection reagent highly sensitive, the present invention adds magnesium sulfate as an enzyme activator to the system; in order to make the detection reagent stable for a long time, the present invention dries the second solution into a powder, and heat-seals it together with a desiccant in an opaque aluminum foil bag for storage.

[0019] Another object of the present invention is to provide a method for using the above-mentioned Helicobacter pylori detection reagent, which comprises the following steps: using a disposable sterile sampling swab to brush along the gingival crevice for 2 weeks (see Figure 1yellow track), obtain an oral sample; use purified water to dissolve the powdered Helicobacter pylori detection reagent into a liquid reagent; place the cavity sample in the liquid reagent to react for more than 10 minutes, and observe the color change of the liquid reagent; if the liquid reagent does not change color, it is negative; if the liquid reagent turns red or purple, it is positive.

[0020] For the sampling process required by the Helicobacter pylori detection reagent of the present invention, a sterile sampling swab is used to brush along the gingival crevice for 2 weeks. The whole sampling process is relatively gentle and gentle, while the detection reagents on the market require dental plaque samples, and the whole dental plaque sampling process is relatively simple and rough. In addition, the Helicobacter pylori detection reagent of the present invention needs to be dissolved with pure water before use, and then put into the sampled swab (i.e., oral sample), so that the liquid form of the detection reagent is more tolerant to the influence of non-Helicobacter pylori on the oral sample, such as the influence of food pH.

[0021] The present application has the following beneficial effects: the detection reagent provided by the present invention can effectively detect Helicobacter pylori, and compared with other similar products, has better specificity, sensitivity and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a sampling site diagram of the method for using the Helicobacter pylori detection reagent of the present invention;

[0023] Figure 2 It is the test diagram of the test reagent with different concentrations of antibacterial agent added;

[0024] Figure 3 This is the detection diagram at the first minute after adding different concentrations of magnesium sulfate detection reagents;

[0025] Figure 4 This is the detection diagram at 5 minutes after adding different concentrations of magnesium sulfate detection reagents;

[0026] Figure 5 This is the detection diagram at 10 minutes after adding different concentrations of magnesium sulfate detection reagents;

[0027] Figure 6 This is the test graph of the test reagent with different concentrations of nutrients added at the first minute;

[0028] Figure 7 This is the test graph of the test reagent with different concentrations of nutrients added at 3 minutes;

[0029] Figure 8 This is the test graph of the test reagent with different concentrations of nutrients added at 10 minutes;

[0030] Fig. 9This is a commercially available Helicobacter pylori test strip. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with embodiments, but the scope of protection claimed by the present invention is not limited to the scope described in the embodiments.

[0032] Currently, the Helicobacter pylori detection test strips and Helicobacter pylori detection reagents available on the market are mostly for the detection of oral samples, but the test results are inaccurate. It is speculated that the test strips may be intolerant to the pH of oral samples and have low reaction sensitivity, resulting in errors in the test results. In addition, there are many false positive and false negative problems in the test strips sold on the market. However, the present invention has higher specificity, sensitivity and stability because the pH value of the reagent is 7.6, the reagent contains components such as antibacterial agents, enzyme activators, nutrients, and the reagent tube is in a dry state.

[0033] Example 1: Screening of different concentrations of antibacterial agents to test the specificity of the detection reagent

[0034] (1) Preparation of antibacterial agent: Add 10 g of vancomycin, 8 g of trimethylolpropane, and 5 g of amphotericin B into a measuring cylinder, add purified water to make up to 100 ml, and mix evenly to obtain the antibacterial agent.

[0035] (2) Preparation of buffer solution: Add 1 g sodium chloride, 0.04 g potassium chloride, 1 g potassium dihydrogen phosphate, 1 g disodium hydrogen phosphate, and 0.5 g sodium hydroxide into a measuring cylinder, add pure water to make up to 100 ml, and mix evenly.

[0036] (3) Preparation of the test stock solution: Add 10 g urea and 0.01 g phenol red into a measuring cylinder, add pure water to make up to 100 ml, and mix evenly.

[0037] (4) Preparation of nutrient solution: Add 5 g of glucose and 5 g of tris(hydroxymethyl)aminomethane (Tr is) into a measuring cylinder, add pure water to make up to 100 ml, and mix evenly.

[0038] (5) Preparation of detection reagents containing different concentrations of antibacterial agents: 20 g of buffer, Xg of antibacterial agent, 5 mM magnesium sulfate, 2 g of nutrient agent, and 20 g of detection mother solution were added to a measuring cylinder, and the volume was fixed to 100 ml with pure water, and the mixture was uniformly mixed. Among them, Xg was set to 0 g, 0.8 g, 1.6 g, and 3.2 g, respectively.

[0039] (6) Sample addition: 1 uL of the mixed bacterial solution (a mixed bacterial solution formed by Aspergillus niger, Candida albicans, and Proteus mirabilis) was added to the detection reagent containing different concentrations of antibacterial agents prepared above.

[0040] (7) The results show that the test reagents do not change color when the antibacterial agent is added in an amount of 1.6% or more, so the amount of antibacterial agent added is 1.6%. The test results are as follows Figure 2 shown.

[0041] Example 2: Screening different concentrations of magnesium sulfate to test the sensitivity of the detection reagent

[0042] (1) The preparation methods of the antibacterial agent, buffer solution, detection mother solution and nutrient solution are the same as those in Example 1.

[0043] (2) Preparation of detection reagents containing different concentrations of magnesium sulfate: 20 g of buffer, 1.6 g of antibacterial agent, YmM magnesium sulfate, 2 g of nutrient agent, and 20 g of detection mother solution were added to a measuring cylinder, and the volume was fixed to 100 ml with pure water, and the mixture was evenly mixed. Among them, YmM was set to 2mM, 5mM, and 10mM, respectively.

[0044] (3) Sample addition: 1 uL of urease solution (0.025 U / uL) was added to the detection reagents containing different concentrations of magnesium sulfate prepared above.

[0045] (4) The results showed that at the first minute, the three groups did not change color; at the fifth minute, the 5mM group changed color, while the 2mM group and the 10mM group did not change color; at the tenth minute, all three groups changed color. Figures 3 to 5 shown.

[0046] Example 3: Screening of nutrients of different concentrations to test the sensitivity of the detection reagent

[0047] (1) The preparation methods of the antibacterial agent, buffer solution, detection mother solution and nutrient solution are the same as those in Example 1.

[0048] (2) Preparation of detection reagents containing different concentrations of nutrient agents: Add 20 g of buffer, 1.6 g of antibacterial agent, 5 mM magnesium sulfate, Zg of nutrient agent, and 20 g of detection mother solution into a measuring cylinder, add pure water to make the volume to 100 ml, and mix evenly. Among them, Zg is set to 0 g, 5 g, and 10 g, respectively.

[0049] (3) Adding samples: Add 1 uL of Helicobacter pylori solution to the detection reagents containing different concentrations of nutrients prepared above.

[0050] (4) The results show that: at the first minute, the three groups did not change color; at the third minute, the 0g group did not change color, and the 5g group and the 10g group both changed color; at the tenth minute, the three groups all changed color; that is, when the nutrient is 5g (5wt%), the sensitivity can be improved. The test results are as follows: Figures 6 to 8 shown.

[0051] Example 4: Testing the stability of the detection reagent in dry and liquid states after 12 months of storage

[0052] (1) The preparation methods of the antibacterial agent, buffer solution, detection mother solution and nutrient solution are the same as those in Example 1.

[0053] (2) Preparation of detection reagent: Add 20 g of buffer solution, 1.6 g of antibacterial agent, 5 mM magnesium sulfate, 5 g of nutrient agent, and 20 g of detection mother solution into a measuring cylinder, add pure water to make up to 100 ml, and mix evenly.

[0054] (3) The detection reagents prepared above were divided into two groups. One group was directly heat-sealed in a light-proof aluminum foil bag; the other group was placed in a forced air drying oven at 55°C and dried to powder form, and then heat-sealed in a light-proof aluminum foil bag with a desiccant; both groups of detection reagents were stored at room temperature away from light for 12 months.

[0055] (4) After 12 months, the liquid group was directly inspected for appearance and performance test; the dry group was dissolved in 100 μl of pure water and then inspected for appearance and performance test.

[0056] (5) More specifically, the method for performance testing of step (4) is to add 1 u l of positive quality control and 1 u l of negative quality control to the test reagent respectively; the positive quality control is Helicobacter pylori solution, and the negative quality control is a mixed bacterial solution composed of Aspergillus niger, Candida albicans, and Proteus mirabilis.

[0057] (6) After 10 minutes, check whether the test liquid has changed color. It should be noted that the liquid group is already red before the reaction, so whether it changes color is used as the comprehensive performance evaluation standard.

[0058] (7) Each group was tested 5 times, and the results are shown in Table 1 below:

[0059] Table 1 Stability of detection reagents stored in different states

[0060]

[0061]

[0062] Example 5: Comparison of the detection effect of the detection reagent of the present invention and commercially available test strips The test was performed according to the method shown in Table 2 below, and the results are shown in Table 3.

[0063] Table 2 Comparison of test methods

[0064]

[0065]

[0066] Table 3 Comparison test results

[0067]

[0068] It can be seen from Tables 1 to 4 that the detection reagent provided by the present invention can effectively detect Helicobacter pylori, and has better specificity, sensitivity and stability than other similar products.

[0069] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A Helicobacter pylori detection reagent based on oral sampling, characterized in that: The preparation method comprises the following steps: S1: Evenly mix the buffer solution, the antibacterial agent, the enzyme activator and the nutrient agent to obtain a first solution; S2: adding the detection mother solution to the first solution and mixing them evenly to obtain a second solution; S3: transferring the second solution into a reagent tube and drying it into a powder to obtain a test powder; S4: heat-sealing the detection powder and the desiccant in a light-proof aluminum foil bag to obtain the Helicobacter pylori detection reagent; The pH value of the second solution is 7.6; The buffer solution is composed of the following components in percentage by mass: 0.5-1.5% sodium chloride, 0.02-0.05% potassium chloride, 0.5-1.5% potassium dihydrogen phosphate, 0.5-1.5% disodium hydrogen phosphate, 0.2-0.6% sodium hydroxide and the balance pure water; The antibacterial agent is composed of the following components in percentage by mass: 10-12% vancomycin, 8-10% trimethylolpropane, 4-6% amphotericin B and the balance of pure water; the amount of the antibacterial agent added to the Helicobacter pylori detection reagent is 1.6-3.2%; The enzyme activator is magnesium sulfate, and its content is 2-10 mM; The nutrient is composed of the following components in percentage by mass: 4-6% glucose, 4-6% tris(hydroxymethyl)aminomethane and the balance pure water; The detection mother solution is composed of the following components in percentage by mass: 10-12% urea, 0.01-0.02% phenol red and the balance pure water; In step S3, the second solution is dried to powder at 55-60°C.

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