Helicobacter pylori staining solution for rapid detection

Abstract

The invention relates to a helicobacter pylori staining solution for rapid detection, which consists of a staining solution and a diluent, the staining solution comprises the following components in percentage by weight: 0.4-0.6% of phenol red, 30-40% of ethanol and 60-70% of purified water, and the pH range of the staining solution is 6.4-6.6. The diluent comprises the following components in percentage by weight: 10%-14% of urea, 0.01%-0.03% of Triton X-100 and the balance of purified water, and the pH range of the diluent is 6.4-6.6. The volume ratio of the staining solution to the diluent is (1: 4.5)-(1: 5.5). The staining solution is simple and rapid in operation process, and one-step staining is achieved, so that personnel operation is facilitated; the reaction is efficient, color development can be completed within 5-10 minutes, and the method is suitable for rapid detection of scenes and convenient to popularize; the staining solution is high in staining efficiency, easy to observe, low in staining cost and high in market competitiveness; the staining solution is stable in performance, easy to transport and store at normal temperature and beneficial to long-term use of reagents; the staining solution is safe and non-toxic in formula, and is safe to operators and environment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of dyeing solution, and particularly relates to a Helicobacter pylori dyeing solution for rapid detection. BACKGROUND

[0002] With the increasing requirements of clinical detection accuracy and efficiency of Helicobacter pylori, dyeing technologies based on different principles have gradually developed. On the one hand, the maturity of technologies such as immunofluorescence technology, methylene blue dyeing technology and enzyme reaction coloration provides new ideas for the research and development of dyeing solution; on the other hand, the defects of traditional dyeing solution in stability, color development contrast and operation convenience need to be solved.

[0003] Helicobacter pylori can produce urease and can decompose urea to produce ammonia, which can increase the pH value of the environment. Some dyeing solutions use phenol red as an acid-base indicator. When Helicobacter pylori exists in the sample, the urease produced by Helicobacter pylori decomposes the urea in the dyeing solution, and the ammonia produced increases the pH value, and the phenol red changes from yellow to red, thereby realizing the detection of Helicobacter pylori.

[0004] Helicobacter pylori (Hp for short) is a spiral-shaped, micro-anaerobic and strict bacteria. Helicobacter pylori is the main pathogenic factor of chronic gastritis, peptic ulcer (gastric ulcer, duodenal ulcer), and is closely related to serious diseases such as gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma (MALT lymphoma). Long-term infection of Helicobacter pylori can cause repeated damage and repair of gastric mucosa, and then gradually lead to mucosal atrophy, intestinal metaplasia and dysplasia. The accumulation of this series of precancerous lesions can significantly increase the risk of gastric cancer. Timely detection and eradication of Helicobacter pylori can significantly reduce the recurrence rate of peptic ulcer (from 50%-70% per year to less than 5%), and can prevent the progression of precancerous lesions of gastric mucosa, even reverse some early lesions, and effectively prevent the occurrence of gastric cancer.

[0005] Traditional Helicobacter pylori detection relies on bacterial culture, which needs to be cultured in a specific culture medium for 3-7 days, and the culture conditions are harsh (micro-aerobic environment), which is difficult to meet the clinical rapid diagnosis demand. The existing technology has a faster operation reaction speed, but the sample and the diluent need to be added to the dyeing solution in multiple steps, and the operation process is complex. For the clinic, when a large number of samples need to be detected, the tedious steps will prolong the detection period and reduce the efficiency; for individuals and non-professionals, it will reduce the convenience and fault tolerance of detection. SUMMARY

[0006] To address the aforementioned issues, a staining solution for rapid detection of Helicobacter pylori has been proposed. The principle is that urease produced by Helicobacter pylori decomposes urea to produce ammonia, raising the ambient pH. Phenol red (an acid-base indicator) changes from yellow to red under alkaline conditions, and the presence of infection can be determined within 5-10 minutes through this color change, significantly shortening the detection time and making it suitable for rapid clinical screening. This staining solution can be directly used for rapid detection of fresh human saliva samples or gastric mucosal tissue, requiring no complex equipment or slide preparation. Results can be determined simply by observing the color change with the naked eye, reducing reliance on laboratory conditions and operator skills, making it suitable for primary healthcare institutions or rapid point-of-care testing.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows.

[0008] A staining solution for rapid detection of Helicobacter pylori comprises a staining solution and a diluent. The staining solution consists of phenol red, anhydrous ethanol, and purified water, while the diluent consists of urea, Triton X-100, and purified water.

[0009] Furthermore, the staining solution contains, by weight percentage, 0.4%–0.6% phenol red, 30%–40% ethanol, and 60%–70% purified water, with a pH range of 6.4–6.6.

[0010] Furthermore, the staining solution contains, by weight percentage, 0.4% phenol red, 30% ethanol, and the remainder purified water, with a pH of 6.5.

[0011] Furthermore, the staining solution contains, by weight percentage, 0.5% phenol red, 30% ethanol, and the remainder purified water, with a pH of 6.5.

[0012] Furthermore, the staining solution contains, by weight percentage, 0.6% phenol red, 40% ethanol, and the remainder purified water, with a pH of 6.5.

[0013] Furthermore, the diluent contains, by weight percentage, 10%–14% urea, 0.01%–0.03% Triton X-100, and the remainder is purified water, with a pH range of 6.4–6.6.

[0014] Furthermore, the diluent consists of 10% urea, 0.01% Triton X-100, and the remainder purified water by weight, with a pH of 6.5.

[0015] Furthermore, the diluent consists of 14% urea, 0.03% Triton X-100, and the remainder purified water by weight, with a pH of 6.5.

[0016] Furthermore, the volume ratio of staining solution to diluent is 1:4.5 to 1:5.5.

[0017] The preparation method of the above Helicobacter pylori staining solution is as follows: Weigh each raw material according to the above formula, add phenol red to ethanol and purified water, stir until completely dissolved, and adjust the pH with sodium hydroxide to obtain the staining solution. Add urea and Triton X-100 to purified water, stir thoroughly, and adjust the pH with hydrochloric acid to obtain a diluted solution. Add the diluted solution to the staining solution, mix well, seal in a dark place, and store at room temperature.

[0018] The beneficial effects of this invention are: (1) The operation process is simple and quick, with a one-step staining step, which is convenient for personnel to operate; (2) The reaction is highly efficient, and the color development can be completed in 5-10 minutes, which is suitable for rapid detection scenarios and easy to promote; (3) The staining solution has high staining efficiency, is easy to observe, has low staining cost, and has strong market competitiveness; (4) The staining solution has stable performance, is easy to transport and store at room temperature, and is conducive to long-term use of reagents; (5) The staining solution formula is safe and non-toxic, and is safe for operators and the environment. Attached Figure Description

[0019] Figure 1 This is the distribution curve of the number of Helicobacter pylori positive samples under different incubation times in Example 1 of the present invention. Detailed Implementation

[0020] The specific embodiments of the present invention will be described below in conjunction with examples to provide a better understanding of the present invention.

[0021] Example 1

[0022] The Helicobacter pylori staining solution for rapid detection in this embodiment consists of a staining solution and a diluent. The staining solution comprises, by weight percentage, 0.4% phenol red, 30% ethanol, and the remainder purified water, with a pH of 6.5. The diluent comprises, by weight percentage, 10% urea, 0.01% Triton X-100, and the remainder purified water, with a pH of 6.5. The preparation method of the above Helicobacter pylori staining solution is as follows: Weigh each raw material according to the above formula; add phenol red to ethanol and purified water, stir until completely dissolved, and adjust the pH with sodium hydroxide to obtain the staining solution. Add urea and Triton X-100 to the purified water, stir thoroughly, and adjust the pH with hydrochloric acid to obtain the diluent. Add 4.5 parts of the diluent to 1 part of the staining solution, mix well, seal away from light, and store at room temperature.

[0023] Example 2

[0024] The Helicobacter pylori staining solution for rapid detection in this embodiment consists of a staining solution and a diluent. The staining solution comprises, by weight percentage, 0.5% phenol red, 30% ethanol, and the remainder purified water, with a pH of 6.5. The diluent comprises, by weight percentage, 10% urea, 0.01% Triton X-100, and the remainder purified water, with a pH of 6.5. The preparation method of the above Helicobacter pylori staining solution is as follows: Weigh each raw material according to the above formula; add phenol red to ethanol and purified water, stir until completely dissolved, and adjust the pH with sodium hydroxide to obtain the staining solution. Add urea and Triton X-100 to the purified water, stir thoroughly, and adjust the pH with hydrochloric acid to obtain the diluent. Add 5 parts of the diluent to 1 part of the staining solution, mix well, seal away from light, and store at room temperature.

[0025] Example 3

[0026] The Helicobacter pylori staining solution for rapid detection in this embodiment consists of a staining solution and a diluent. The staining solution comprises, by weight percentage, 0.5% phenol red, 30% ethanol, and the remainder purified water, with a pH of 6.5. The diluent comprises, by weight percentage, 14% urea, 0.03% Triton X-100, and the remainder purified water, with a pH of 6.5. The preparation method of the above Helicobacter pylori staining solution is as follows: Weigh each raw material according to the above formula; add phenol red to ethanol and purified water, stir until completely dissolved, and adjust the pH with sodium hydroxide to obtain the staining solution. Add urea and Triton X-100 to purified water, stir thoroughly, and adjust the pH with hydrochloric acid to obtain the diluent. Add 5 parts of the diluent to 1 part of the staining solution, mix well, seal away from light, and store at room temperature.

[0027] Example 4

[0028] The Helicobacter pylori staining solution for rapid detection in this embodiment consists of a staining solution and a diluent. The staining solution comprises, by weight percentage, 0.6% phenol red, 40% ethanol, and the remainder purified water, with a pH of 6.5. The diluent comprises, by weight percentage, 14% urea, 0.03% Triton X-100, and the remainder purified water, with a pH of 6.5. The preparation method of the above Helicobacter pylori staining solution is as follows: Weigh each raw material according to the above formula; add phenol red to ethanol and purified water, stir until completely dissolved, and adjust the pH with sodium hydroxide to obtain the staining solution. Add urea and Triton X-100 to purified water, stir thoroughly, and adjust the pH with hydrochloric acid to obtain the diluent. Add 5.5 parts of the diluent to 1 part of the staining solution, mix well, seal away from light, and store at room temperature.

[0029] Clinical trials:

[0030] Fifty patients with gastritis from Qingdao Yasi Biotechnology Co., Ltd. were randomly selected and designated as experimental groups S1 to S50. A negative control group S0 (sterile saline solution simulating a sample without *Helicobacter pylori*) and a positive control group SP (urease at a concentration of 0.5 U / mL) were also established. Fresh saliva was collected and stained using the staining solution prepared in Example 1. Each group of samples was tested in triplicate, with three parallel tests performed for each sample. The color change in each test tube was observed and recorded within 30 minutes and graded according to the following criteria:

[0031] Level 0: Yellow (same as initial color) → Negative

[0032] Level 1: Orange-red (slight discoloration) → Negative

[0033] Level 2: Light pink (obvious discoloration) → Positive

[0034] Level 3: Deep pink (drastic color change) → Strong positive

[0035] The positive control group must achieve a grade 2 or higher color change within 30 minutes, while the negative control group must remain at grade 0; otherwise, the test is invalid. A positive result is achieved in at least two out of three replicates within 30 minutes, indicating a grade 2 or higher change.

[0036] Table 1. Color changes and judgment results for each sample within 30 minutes.

[0037]

[0038]

[0039] As observed in Table 1, among the 50 patients suffering from gastritis, 19 were positive after staining with Helicobacter pylori staining solution. The positive rate was: positive rate = number of positive samples / total number of samples × 100% = 19 / 50 × 100% = 38%.

[0040] Based on Table 1, plot the distribution of the number of Helicobacter pylori positive samples under different incubation times, as shown in the figure. Figure 1 The results showed that the number of positive samples gradually increased when the reaction time was 0–10 min, with the fastest Hp reaction rate. The number of positive samples tended to reach equilibrium at 10 min, and the increase slowed down after 10 min, with a significant decrease in Hp activity. Therefore, 10 min is the optimal time for visual observation of color development.

[0041] Consistency check

[0042] In the interpretation of the results of the Helicobacter pylori staining solution method, 19 positive patients underwent C... 14 Further testing was conducted using the urea breath test (gold standard), and the results are shown in Table 2.

[0043] Table 2. Helicobacter pylori staining solution, staining method, and C...14 Urea breath test (gold standard) comparison

[0044]

[0045] 1. Accuracy

[0046] The proportion of all test results that are consistent with the gold standard (including true positive and true negative results).

[0047]

[0048] It has a high accuracy in reflecting the overall results of Helicobacter pylori staining solution and shows high consistency with the gold standard results.

[0049] 2. Sensitivity (true positive rate)

[0050] The proportion of samples that tested positive using the gold standard and were correctly detected by the testing method.

[0051]

[0052] This indicates that the Helicobacter pylori staining solution has a high ability to identify positive samples and a low rate of missed diagnoses.

[0053] 3. Specificity (true negative rate)

[0054] The proportion of samples that tested negative using the gold standard that were correctly detected by the testing method.

[0055]

[0056] This indicates that the Helicobacter pylori staining solution has a high ability to identify negative samples and a low misdiagnosis rate.

[0057] 4. Positive Predictive Value (PPV)

[0058] The proportion of samples that are actually positive (confirmed by the gold standard) among those detected positive by the test method.

[0059]

[0060] The positive results of Helicobacter pylori staining solution reflect a high degree of confidence, meaning that the probability of a "true positive" result is high.

[0061] 5. Negative Predictive Value (NPV)

[0062] The proportion of samples that tested negative by the test method that were truly negative (confirmed by the gold standard).

[0063]

[0064] The high reliability of negative results from Helicobacter pylori staining solution indicates a high probability of "true negative" results.

[0065] 6. Kappa coefficient (consistency test)

[0066] The consistency of the results from the two methods is considered if κ ≥ 0.6, indicating good consistency and high reliability.

[0067] formula:

[0068] in: (Actual consistency rate)

[0069] (Expected Consistency Rate)

[0070] The result is: This means that the Helicobacter pylori staining solution has a high degree of consistency with the gold standard.

[0071] The above indicators can be used to comprehensively evaluate the Helicobacter pylori staining solution relative to C. 14 The accuracy of the urea breath test confirms its feasibility as an alternative detection method.

[0072] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A staining solution for rapid detection of Helicobacter pylori, comprising a staining solution and a diluent, characterized in that: The staining solution consists of phenol red, anhydrous ethanol, and purified water, while the diluent consists of urea, Triton X-100, and purified water.

2. The staining solution for rapid detection of Helicobacter pylori according to claim 1, characterized in that: The staining solution consists of 0.4%–0.6% phenol red, 30%–40% ethanol, and 60%–70% purified water by weight, with a pH range of 6.4–6.

6.

3. The staining solution for rapid detection of Helicobacter pylori according to claim 2, characterized in that: The staining solution consists of 0.4% phenol red, 30% ethanol, and the remainder is purified water, with a pH of 6.

5.

4. The staining solution for rapid detection of Helicobacter pylori according to claim 2, characterized in that: The staining solution consists of 0.5% phenol red, 30% ethanol, and the remainder is purified water, with a pH of 6.

5.

5. The staining solution for rapid detection of Helicobacter pylori according to claim 2, characterized in that: The staining solution consists of 0.6% phenol red, 40% ethanol, and the remainder is purified water, with a pH of 6.

5.

6. The staining solution for rapid detection of Helicobacter pylori according to claim 1, characterized in that: The diluent contains, by weight percentage, 10%–14% urea, 0.01%–0.03% Triton X-100, and the remainder is purified water, with a pH range of 6.4–6.

6.

7. The staining solution for rapid detection of Helicobacter pylori according to claim 6, characterized in that: The diluent consists of 10% urea, 0.01% Triton X-100, and the remainder purified water by weight, with a pH of 6.

5.

8. The staining solution for rapid detection of Helicobacter pylori according to claim 6, characterized in that: The diluent consists of 14% urea, 0.03% Triton X-100, and the remainder purified water by weight, with a pH of 6.

5.

9. The staining solution for rapid detection of Helicobacter pylori according to claim 1, characterized in that: The volume ratio of the staining solution to the diluent is 1:4.5 to 1:5.

5.

10. The staining solution for rapid detection of Helicobacter pylori according to any one of claims 1-9, characterized in that: The preparation method of the above Helicobacter pylori staining solution is as follows: Weigh each raw material according to the above formula, add phenol red to ethanol and purified water, stir until completely dissolved, and adjust the pH with sodium hydroxide to obtain the staining solution. Add urea and Triton X-100 to purified water, stir thoroughly, and adjust the pH with hydrochloric acid to obtain a diluted solution. Add the diluted solution to the staining solution, mix well, seal in a dark place, and store at room temperature.

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