Standard colorimetric card and kit for rapid semi-quantitative detection of Helicobacter pylori in saliva

Abstract

The present invention discloses a rapid semi-quantitative detection method and kit for Helicobacter pylori in saliva based on an indicator method. The method is suitable for detecting Helicobacter pylori infection and can provide semi-quantitative indication through color difference to preliminarily determine the degree of Helicobacter pylori infection. The method uses an acid-base indicator that can replace phenol red for Helicobacter pylori urease detection. Using real saliva containing Helicobacter pylori as a detection model, a semi-quantitative indicator method for Helicobacter pylori in saliva is established. The method has high sensitivity, simple operation, low cost, and can visually and semi-quantitatively determine the Helicobacter pylori content in saliva.

Description

Technical Field

[0001] The present invention relates to a method for rapid semi-quantitative detection of Helicobacter pylori in saliva using a mixed acid-base indicator, and also relates to a kit for rapid semi-quantitative detection of Helicobacter pylori in saliva using the mixed acid-base indicator. Background Art

[0002] As the knowledge of the dangers of Helicobacter pylori (Hp) has spread to thousands of households, more and more people have realized the high incidence and serious pathogenicity of Helicobacter pylori infection, as well as the importance of early diagnosis of Helicobacter pylori in later treatment. Currently, there are four commonly used detection methods for Helicobacter pylori in clinical practice: rapid urease (RUT), 13 C breath test ( 13 C-UBT), 14 C breath test ( 14 C-UBT) and blood Hp-IgG method. The basic principle of the first three methods is urease detection. Urease, as a marker for Helicobacter pylori detection, has always been of great value in the detection of Hp infection. However, the breath test requires special equipment, especially 14 C-UBT involves radiation and is unsuitable for children and pregnant women, while endoscopic sampling for RUT or histological examination is limited by patient compliance. Existing methods generally suffer from shortcomings such as complex testing procedures, inconvenient operation, and non-intuitive test results.

[0003] The phenol red indicator method (also known as the pH indicator method) is a classic urease detection method. It utilizes the urease enzyme's ability to catalyze the decomposition of urea to produce ammonia, which increases the pH of the system and subsequently changes the color of the phenol red in the system, thereby diagnosing H. pylori infection. Studies have shown that H. pylori can colonize the oral cavity, and gastric H. pylori is closely related to oral H. pylori infection. Saliva is an in vitro specimen and is harmless to the human body. Therefore, detecting H. pylori urease in saliva is convenient and has guiding significance for disease diagnosis.

[0004] Patented or commercially available oral Helicobacter pylori self-test strips are mostly made with phenol red and perform only qualitative testing. However, the pH of saliva in healthy individuals is generally between 6.0 and 7.9, while the color change range of phenol red is between 6.8 and 8.4, making phenol red an unsuitable indicator for Helicobacter pylori detection in saliva. Furthermore, there is currently very little research on innovative phenol red indicators in this method, or on the application of acid-base indicators to achieve intuitive, semi-quantitative test results. Summary of the Invention

[0005] In order to overcome the shortcomings and deficiencies in the prior art, the purpose of the present invention is to prepare an acid-base indicator that can replace phenol red for Helicobacter pylori urease detection, and to use the indicator to establish a semi-quantitative method for Helicobacter pylori in saliva, thereby providing a rapid, accurate and reliable detection method for Helicobacter pylori.

[0006] Another object of the present invention is to provide a kit for rapid semi-quantitative detection of Helicobacter pylori in saliva using the acid-base indicator.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A standard colorimetric card for rapid semi-quantitative detection of Helicobacter pylori in saliva is prepared by the following steps:

[0009] (1) Preparation of acid-base indicator: Mix bromophenol blue solution, ethyl orange solution, bromocresol green solution, hydrochloric acid and ethanol to prepare the acid-base indicator;

[0010] (2) Detection of Helicobacter pylori in saliva: 150-250 μL of urea-NiCl2 solution was mixed with 50-250 μL of saliva containing Helicobacter pylori, and the mixture was reacted for 20-40 min. Then, 500-1000 μL of the acid-base indicator prepared in step (1) was added. After 30-90 s, the absorbance value was detected at 600 nm after the reaction solution was stabilized.

[0011] (3) Determine the standard curve of Helicobacter pylori: use the concentration of Helicobacter pylori as the horizontal axis and the absorbance as the vertical axis to obtain the standard curve;

[0012] (4) Preparation of Helicobacter pylori standard colorimetric cards: Experiments were conducted using saliva at pH 7.0. The pH values ​​of saliva with different Helicobacter pylori concentrations after reaction with urea-NiCl2 solution were measured, and the color of the acid-base indicator was recorded to prepare standard colorimetric cards corresponding to the initial saliva pH values ​​of 7.0, 6.0, and 6.5.

[0013] Furthermore, the standard colorimetric card for rapid semi-quantitative detection of Helicobacter pylori in saliva mentioned above is prepared according to the following steps: bromophenol blue solution, ethyl orange solution, bromocresol green solution, 0.02 mol / L hydrochloric acid, and 90% ethanol are mixed in a volume ratio of 5:5:10:18:120 to prepare an acid-base indicator.

[0014] Furthermore, in the above-mentioned standard colorimetric card for rapid semi-quantitative detection of Helicobacter pylori in saliva, the urea concentration in the urea-NiCl2 solution is 5wt%-50wt%.

[0015] Furthermore, the above-mentioned standard colorimetric card for rapid semi-quantitative detection of Helicobacter pylori in saliva, step (2) is specifically as follows: weigh 200 μL of urea-NiCl2 solution with a urea concentration of 25 wt%, add 200 μL of saliva containing Helicobacter pylori, react for 30 minutes, add 800 μL of indicator, wait for 30-90 seconds for the reaction solution to stabilize, and then detect the absorbance value at 600 nm.

[0016] Furthermore, the standard colorimetric card for the rapid semi-quantitative detection of Helicobacter pylori in saliva is used. The specific method for determining the standard curve of Helicobacter pylori in step (3) is as follows: the concentration is 0 CFU / mL, 1×10 4 CFU / mL, 2×10 4 CFU / mL, 4×10 4 CFU / mL, 6×10 4 CFU / mL, 8×10 4 CFU / mL, 1×10 5 CFU / mL, 2×10 5 CFU / mL, 4×10 5 CFU / mL, 6×10 5 CFU / mL, 8×10 5 CFU / mL, 1×10 6 CFU / mL, 2×10 6 CFU / mL, 4×10 6 CFU / mL, 6×10 6 CFU / mL, 8×10 6 CFU / mL, 1×10 7 CFU / mL, 2×10 7 CFU / mL, 4×10 7 CFU / mL, 6×10 7 CFU / mL, 8×10 7 CFU / mL, 1×10 8 CFU / mL of Helicobacter pylori standard solution was used to detect Helicobacter pylori of different concentrations using the method described in step (2), the absorbance value at 600 nm was recorded, and a standard curve was drawn.

[0017] Furthermore, the standard colorimetric card for the rapid semi-quantitative detection of Helicobacter pylori in saliva is prepared in step (4) by preparing a standard colorimetric card with a concentration of 0 CFU / mL and 1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8CFU / mL Helicobacter pylori standard solution, use the method described in step (2) to determine the pH value of urea-NiCl2 solution after reacting with saliva for 30 minutes, use 0.5mol / mL NaOH solution to adjust the Hp-free saliva to pH 6.31±0.02, pH 6.37±0.01, pH 6.42±0.01, pH 6.77±0.01, pH 8.37±0.02, pH 9.17±0.01, and add 800μL of indicator respectively, mix well to make colorimetric tube, compare with Pantone color, and record the color number; immerse the cut filter paper in the colorimetric tube, take out after it is completely soaked, dry at 50℃, then wash away the floating color with NaOH solution of corresponding pH value for 5 minutes, dry at 50℃ to make a standard colorimetric card, and compare with the corresponding PANTONE number color to check the color difference.

[0018] The second technical solution provided by the present invention is a rapid semi-quantitative detection kit for Helicobacter pylori in saliva, comprising a box body, wherein the box body is provided with two rapid urease reagent cups, a saliva measuring cup, a soft plastic bottle A containing 1 to 10 mL of urea-nickel chloride solution, a soft plastic bottle B containing 1 to 10 mL of deionized water, a soft plastic bottle C containing 1 to 10 mL of the acid-base indicator prepared according to claim 2, several standard colorimetric cards for rapid semi-quantitative detection of Helicobacter pylori in saliva according to the first technical solution, and a pouch straw.

[0019] Furthermore, in the above-mentioned rapid semi-quantitative detection kit for Helicobacter pylori in saliva, the soft plastic bottle A, the soft plastic bottle B, and the soft plastic bottle are all equipped with a dripper.

[0020] Furthermore, the above-mentioned rapid semi-quantitative detection kit for Helicobacter pylori in saliva comprises three standard colorimetric cards for rapid semi-quantitative detection of Helicobacter pylori in saliva.

[0021] Compared with the existing methods, the present invention has the following advantages and beneficial effects:

[0022] In addition to being able to simply, non-invasively, quickly and inexpensively determine whether Helicobacter pylori is present by detecting the presence of urease in saliva, the detection method of the present invention can also be used for rapid semi-quantification of Helicobacter pylori in saliva to preliminarily determine the degree of Helicobacter pylori infection. At the same time, the overall reaction is carried out in a closed environment, which is conducive to improving sensitivity, and has good application value and prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the detection effect and standard curve described in the present invention.

[0024] Figure 2 It is the detection effect before and after the optimization described in the present invention.

[0025] Figure 3 This is the technical route for using the kit described in the present invention.

[0026] Figure 4 This is a schematic diagram of the structure of a rapid semi-quantitative detection kit for Helicobacter pylori in saliva provided by the present application. DETAILED DESCRIPTION

[0027] The present invention will be further described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto.

[0028] Example 1

[0029] The present invention provides a standard colorimetric card for rapid semi-quantitative detection of Helicobacter pylori in saliva, comprising the following steps:

[0030] Step 1) Preparation of acid-base indicator

[0031] Prepare the acid-base indicator as follows: Accurately weigh 0.1 g of bromophenol blue powder, dissolve it in 7.15 mL of 0.02 mol / L NaOH solution, dilute to 250 mL with deionized water, and mix thoroughly to obtain a bromophenol blue solution. Accurately weigh 0.4 g of ethyl orange powder, add it to a conical flask, add 250 mL of deionized water, sonicate for 20 minutes, and mix thoroughly to obtain a 0.16% ethyl orange aqueous solution. Accurately weigh 0.1 g of bromocresol green powder, add it to a beaker, dissolve it in 7.45 mL of 0.02 mol / L NaOH solution, dilute to 250 mL with deionized water, and mix thoroughly to obtain a bromocresol green solution.

[0032] Subsequently, the bromophenol blue solution, ethyl orange solution, bromocresol green solution, 0.02 mol / L hydrochloric acid, and 90% ethanol prepared above were uniformly mixed in a volume ratio of 5:5:10:18:120 to prepare the target acid-base indicator.

[0033] Step 2) Detection of Helicobacter pylori in saliva

[0034] 1) Detection of Helicobacter pylori in saliva: scrape freshly cultured Helicobacter pylori, collect the precipitate by centrifugation, take an appropriate amount of the precipitate and dissolve it in PBS, use McFarland turbidimetry to estimate the concentration of Helicobacter pylori, and prepare 21.0×10 9 CFU / mL bacterial solution was diluted with saliva (initial pH value was about 6.0) to prepare the same Helicobacter pylori concentration gradient of 0 CFU / mL, 1×10 4 CFU / mL, 2×10 4 CFU / mL, 4×10 4 CFU / mL, 6×10 4 CFU / mL, 8×10 4 CFU / mL, 1×10 5CFU / mL, 2×10 5 CFU / mL, 4×10 5 CFU / mL, 6×10 5 CFU / mL, 8×10 5 CFU / mL, 1×10 6 CFU / mL, 2×10 6 CFU / mL, 4×10 6 CFU / mL, 6×10 6 CFU / mL, 8×10 6 CFU / mL, 1×10 7 CFU / mL, 2×10 7 CFU / mL, 4×10 7 CFU / mL, 6×10 7 CFU / mL, 8×10 7 CFU / mL, 1×10 8 CFU / mL of saliva samples.

[0035] 2) Weigh 15 g of urea, dissolve it in 100 μmol / L NiCl2 solution, and mix well to prepare a 15 wt% urea-NiCl2 solution.

[0036] 3) Add 200 μL of 15 wt% urea-NiCl2 solution and 200 μL of saliva sample containing Helicobacter pylori into a 1.5 mL Eppendorf tube, mix well, react for 30 minutes, add 800 μL of the acid-base indicator prepared in step 1), mix well, observe and record the color, aspirate 200 μL of the above solution, measure the absorption spectrum, and find that its absorbance at a wavelength of 600 nm (OD 600nm ), the results are given in Figure 2 ,in Figure 2 B is the detection effect before optimization; Figure 2 C is the detection effect after optimization

[0037] Step 3) Determination of Helicobacter pylori standard curve

[0038] 1. Saliva Collection: Rinse mouth with cold water three times for 2 minutes each time. After 1.5 hours, collect saliva from three healthy volunteers. Mix equal amounts of the three saliva aliquots and centrifuge at 10,000 rpm at 4°C for 10 minutes. Collect the supernatant (discard the white, non-transparent mucus at the bottom) and measure the pH, which is approximately 6.0.

[0039] 2. Simulation of saliva sample containing H. pylori: Pipette 1 ml of PBS solution onto the surface of the culture medium, scrape freshly cultured H. pylori, transfer the bacterial solution to a 1.5 mL Eppendorf tube, centrifuge at 8000 rpm, 4°C for 5 minutes to collect the precipitate. Add to PBS and estimate the concentration of H. pylori using McFarland turbidimetry. Prepare 21.0 × 10 9 CFU / mL bacterial solution. Saliva was diluted and treated, and saliva samples with different Helicobacter pylori concentration gradients (0 CFU / mL, 1×10 4 CFU / mL, 2×10 4 CFU / mL, 4×10 4 CFU / mL, 6×10 4 CFU / mL, 8×10 4 CFU / mL, 1×10 5 CFU / mL, 2×10 5 CFU / mL, 4×10 s CFU / mL, 6×10 5 CFU / mL, 8×10 5 CFU / mL, 1×10 6 CFU / mL, 2×10 6 CFU / mL, 4×10 6 CFU / mL, 6×10 6 CFU / mL, 8×10 6 CFU / mL, 1×10 7 CFU / mL, 2×10 7 CFU / mL, 4×10 7 CFU / mL, 6×10 7 CFU / mL, 8×10 7 CFU / mL, 1×10 8 CFU / mL).

[0040] 3. Add urea-NiCl2 solution and saliva containing Helicobacter pylori: Take a 1.5mL Eppendorf tube, add 200μL 15% urea-NiCl2 solution, and add 200μL saliva containing Helicobacter pylori, mix well, and react at room temperature for 30 minutes.

[0041] 4. Color development: Add 800 μL of acid-base indicator, mix well, observe and record the color (within 30-90 seconds). Pipette 200 μL / well and measure OD600nm using a microplate reader.

[0042] Draw the standard curve using origin8.5 software, with OD 600nm The vertical axis is the absorbance value of the negative control group (deducted), and the logarithm of the Helicobacter pylori concentration (C) is the horizontal axis. The four-parameter curve is fitted to obtain the standard curve. Figure 1 , where the corresponding concentrations in tubes 1 to 11 are: 1.0×10 8 CFU / mL~1.0×10 6 CFU / mL; the corresponding concentrations in tubes 12 to 21 are: 8.0×10 5 CFU / m~1.0×10 4 CFU / mL22:0 (no Hp) original saliva pH6.0.

[0043] Example 2

[0044] This example compares the applicability of the present invention and the detection effects before and after optimizing the urea dosage at an initial saliva pH of 7.0, and includes the following steps:

[0045] 1. Saliva Collection: Rinse mouth with cold water three times for 2 minutes each time. After 1.5 hours, collect saliva from three healthy volunteers. Mix the three saliva aliquots and centrifuge them at 10,000 rpm at 4°C for 10 minutes. Collect the supernatant (discard the white, non-transparent mucus at the bottom) and measure the pH, which is approximately 7.0.

[0046] 2. Simulation of saliva sample containing H. pylori: Pipette 1 ml of PBS solution onto the surface of the culture medium, scrape freshly cultured H. pylori, transfer the bacterial solution to a 1.5 mL Eppendorf tube, centrifuge at 8000 rpm, 4°C for 5 minutes to collect the precipitate. Add to PBS and estimate the concentration of H. pylori using McFarland turbidimetry. Prepare 21.0 × 10 9 CFU / mL bacterial solution. Saliva was diluted to prepare saliva samples with different Helicobacter pylori concentration gradients (0 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8 CFU / mL).

[0047] 3. Add urea-NiCl2 solution and saliva containing Helicobacter pylori: Take a 1.5mL Eppendorf tube, add 200μL 15% urea-NiCl2 solution, and add 200μL saliva containing Helicobacter pylori, mix well, and react at room temperature for 30 minutes.

[0048] 4. Color development: Add 800 μL of acid-base indicator, mix well, observe and record the color (within 30-90 seconds). Pipette 200 μL / well and use a microplate reader to measure the absorption spectrum at an incident light wavelength (λ) of 600nm to 800nm. Verification found that for saliva with an initial pH of 7.0, the absorbance at a wavelength of 600nm (OD 600nm ) is still statistically significant.

[0049] 5. Compare the test results under the optimal urea dosage: take out 21.0×10 9 CFU / mL bacterial solution was diluted with saliva to prepare two groups of saliva samples with Helicobacter pylori concentration gradients, with the following concentrations: 0 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8 CFU / mL. Add 200 μL of 15% urea-NiCl2 solution to one sample group and 200 μL of 25% urea-NiCl2 solution to the other sample group. Mix thoroughly and allow to react at room temperature for 30 minutes. Add 800 μL of acid-base indicator to both samples, mix thoroughly, and compare the colors (within 30-90 seconds).

[0050] Example 3

[0051] A rapid semi-quantitative detection kit for Helicobacter pylori in saliva, see Figure 4 , including a box body 1, in which are provided two rapid urease reagent cups 2 (two types numbered a and b), a saliva measuring cup 3, for the convenience of sampling, the saliva measuring cup is a wide-mouth plastic cup, a soft plastic bottle A containing 1 to 10 mL of urea-nickel chloride solution, a soft plastic bottle B containing 1 to 10 mL of deionized water, a soft plastic bottle C containing 1 to 10 mL of the acid-base indicator prepared in Example 1, three standard colorimetric cards 5 for the rapid semi-quantitative detection of Helicobacter pylori in saliva as described in Example 1, and a capsule straw 4.

[0052] The soft plastic bottle A, soft plastic bottle B and soft plastic bottle are all provided with drippers.

[0053] The specific implementation of the kit includes the following steps, see Figure 3 :

[0054] 1. Saliva collection: Rinse your mouth with cold boiled water three times, each time for 2 minutes. After rinsing for 1.5 hours, use a saliva measuring cup to collect 0.5-1 mL of saliva. Blood-containing saliva is not recommended.

[0055] 2. Test: Add 5 drops of urea-nickel chloride solution to the rapid urease reagent cup a and 5 drops of deionized water to the rapid urease reagent cup b. Then, add 5 drops of saliva to both the rapid urease reagent cup a and the rapid urease reagent cup b (try to aspirate the transparent and clear portion), mix well, and let it stand for 30 minutes. Then, add the indicator to the rapid urease reagent cup a and the rapid urease reagent cup b to the scale, mix well, and observe the color change (within 30 to 90 seconds). If there is a significant color difference between the rapid urease reagent cup a and the rapid urease reagent cup b, the test result is positive, indicating that Helicobacter pylori is present in the mouth and H. pylori infection is present. If there is no color difference, the test result is negative and H. pylori infection is not present.

[0056] 3. Colorimetry: Compare the color of the rapid urease reagent cup b with the color in the control column on the colorimetric card, select the standard colorimetric card with the closest colors to the two, and then use the standard colorimetric card to compare the color of the rapid urease reagent cup a for semi-quantitative reading to obtain the H. pylori content in the saliva sample.

[0057] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A standard colorimetric card for rapid semi-quantitative detection of Helicobacter pylori in saliva, characterized in that: Prepared by the following steps: (1) Preparation of acid-base indicator: Mix bromophenol blue solution, ethyl orange solution, bromocresol green solution, 0.02 mol / L hydrochloric acid, and 90% ethanol in a volume ratio of 5:5:10:18:120 to prepare an acid-base indicator; (2) Detection of Helicobacter pylori in saliva: Mix 150-250 μL of urea-NiCl2 solution with 50-250 μL of saliva containing Helicobacter pylori, react for 20 min to 40 min, add 500 μL to 1000 μL of the acid-base indicator prepared in step (1), wait for 30-90 s until the reaction solution stabilizes, and then detect the absorbance at 600 nm; (3) Determine the standard curve of Helicobacter pylori: use the concentration of Helicobacter pylori as the horizontal axis and the absorbance as the vertical axis to obtain the standard curve; (4) Preparation of Helicobacter pylori standard colorimetric card: Experiments were conducted using saliva at pH 7.

0. The pH of saliva with different Helicobacter pylori concentrations after reaction with urea-NiCl2 solution was measured, and the color of the acid-base indicator was recorded to prepare standard colorimetric cards corresponding to the initial saliva pH of 7.0, pH 6.0, and pH 6.

5.

2. The standard colorimetric card for rapid semi-quantitative detection of Helicobacter pylori in saliva according to claim 1, characterized in that: The urea concentration in the urea-NiCl2 solution is 5wt%-50wt%.

3. The standard colorimetric card for rapid semi-quantitative detection of Helicobacter pylori in saliva according to claim 1, characterized in that: Step (2) is specifically as follows: weigh 200 μL of urea-NiCl2 solution with a urea concentration of 25 wt%, add 200 μL of saliva containing Helicobacter pylori, react for 30 minutes, add 800 μL of indicator, wait for 30-90 seconds for the reaction solution to stabilize, and then detect the absorbance value at 600 nm.

4. The standard colorimetric card for rapid semi-quantitative detection of Helicobacter pylori in saliva according to claim 1, characterized in that: The specific method for determining the standard curve of Helicobacter pylori in step (3) is as follows: the concentration is 0 CFU / mL, 1×10 4 CFU / mL, 2×10 4 CFU / mL, 4×10 4 CFU / mL, 6×10 4 CFU / mL, 8×10 4 CFU / mL, 1×10 5 CFU / mL, 2×10 5 CFU / mL, 4×10 5 CFU / mL, 6×10 5 CFU / mL, 8×10 5 CFU / mL, 1×10 6 CFU / mL, 2×10 6 CFU / mL, 4×10 6 CFU / mL, 6×10 6 CFU / mL, 8×10 6 CFU / mL, 1×10 7 CFU / mL, 2×10 7 CFU / mL, 4×10 7 CFU / mL, 6×10 7 CFU / mL, 8×10 7 CFU / mL, 1×10 8 CFU / mL of Helicobacter pylori standard solution was used to detect Helicobacter pylori of different concentrations using the method described in step (2), and the absorbance value at 600 nm was recorded to draw a standard curve.

5. The standard colorimetric card for rapid semi-quantitative detection of Helicobacter pylori in saliva according to claim 1, characterized in that: The specific method of preparing the Helicobacter pylori standard colorimetric card in step (4) is as follows: prepare a concentration of 0 CFU / mL, 1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8 CFU / mL Helicobacter pylori standard solution, the method described in step (2) is used to determine the pH value of urea-NiCl2 solution after reacting with saliva for 30 minutes, and Hp-free saliva is prepared with 0.5mol / mL NaOH solution to pH 6.31±0.02, pH 6.37±0.01, pH 6.42±0.01, pH 6.77±0.01, pH 8.37±0.02, pH 9.17±0.01, and 800μL of indicator is added respectively, mixed and made into colorimetric tubes, compared with Pantone color, and the color number is recorded; the cut filter paper is immersed in the colorimetric tube, taken out after it is completely soaked, dried at 50℃, and then washed with NaOH solution of corresponding pH value for 5 minutes to remove the floating color, dried at 50℃, made into a standard colorimetric card, and compared with the corresponding PANTONE color number to check the color difference.

6. A rapid semi-quantitative detection kit for Helicobacter pylori in saliva, comprising a box body, characterized in that: The box body is provided with two rapid urease reagent cups, a saliva measuring cup, a soft plastic bottle A containing 1 to 10 mL of urea-nickel chloride solution, a soft plastic bottle B containing 1 to 10 mL of deionized water, a soft plastic bottle C containing 1 to 10 mL of an acid-base indicator, several standard colorimetric cards for rapid semi-quantitative detection of Helicobacter pylori in saliva according to any one of claims 1 to 5, and a pipette with a capsule; The acid-base indicator is prepared by uniformly mixing bromophenol blue solution, ethyl orange solution, bromocresol green solution, 0.02 mol / L hydrochloric acid and 90% ethanol in a volume ratio of 5:5:10:18:

120.

7. The rapid semi-quantitative detection kit for Helicobacter pylori in saliva according to claim 6, wherein the soft plastic bottle A, soft plastic bottle B and soft plastic bottle are all provided with a dripper.

8. The kit for rapid semi-quantitative detection of Helicobacter pylori in saliva according to claim 6, wherein the kit comprises three standard colorimetric cards for rapid semi-quantitative detection of Helicobacter pylori in saliva.

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