A kit for detecting purine substances and its application

Abstract

The present invention provides a kit for detecting or assisting in the detection of purine substances, comprising reagent 1, reagent 2, and reagent 3. Reagent 1 is a color developer, reagent 2 is a reaction enzyme, and reagent 3 is a weak acid salt; the reaction enzymes include peroxidase and xanthine oxidase. The kit also includes vitamin C and ascorbic acid enzyme; the vitamin C and ascorbic acid enzyme are packaged separately. The kit of the present invention is rapid and intuitive when detecting a sample to be tested; the kit of the present invention is used to detect purine substances without requiring any instruments or professionally trained personnel to obtain qualitative or semi-quantitative results; when the kit of the present invention is used for detection, even when the sample volume is large, the operation is not restricted, and the kit is easy to operate, improving the user experience.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a kit for detecting purine substances and applications thereof. Background Art

[0002] Xanthine / hypoxanthine is a purine base widely distributed in the organs and body fluids of humans and other organisms. Its normal physiological concentration is 0.5-2.5 μmol / L in serum and 40-160 μmol / L in urine. Uric acid is normally generated by xanthine oxidase (XO) and excreted in urine. Changes in the concentrations of xanthine, hypoxanthine, and uric acid in the human body can directly reflect the state of the body's immune and metabolic functions, and can indicate diseases related to purine metabolism.

[0003] Accumulation of xanthine in the body can easily develop into xanthinuria, which can eventually lead to renal failure. After taking xanthine oxidase inhibitors such as allopurinol and febuxostat, xanthine crystals have been found in the kidneys and ureters, reminding patients to monitor their kidney function and xanthine levels in urine while taking the drug (information source: febuxostat application materials). In addition, in the treatment of hydrocephalus, cerebrospinal fluid (CSF) xanthine / hypoxanthine levels have been used as treatment guidance indicators and disease progression markers.

[0004] The food industry places high demands on the freshness of meat. After fish die, the nucleotides in their bodies are degraded into hypoxanthine and xanthine, whose levels increase with storage time. Therefore, the xanthine content in fish can be used to assess its freshness. In short, xanthine testing is crucial in both the food and medical fields.

[0005] Currently, conventional detection methods such as high-performance liquid chromatography, spectrophotometry, chemiluminescence, and capillary electrophoresis can detect hypoxanthine and xanthine with high sensitivity and selectivity. However, these detection methods are very cumbersome and require relatively expensive equipment and professional and skilled technicians to operate, which increases the cost and time of detection.

[0006] Commercially available XOD activity detection kits are mostly for scientific research purposes, primarily using microplate readers to measure xanthine content. This is complex and has a low throughput. Biochemical analyzer-based assays, on the other hand, are easily accessible and offer high throughput for clinical testing. Furthermore, the development of direct colorimetric assays further simplifies the process, allowing non-professionals to easily determine xanthine levels without time or location constraints.

[0007] Therefore, it is very necessary to develop a method for detecting xanthine that is fast, intuitive, and stable, which can meet the needs of high-throughput quantitative detection such as biochemical analyzers, as well as qualitative and semi-qualitative detection for home portable use. Summary of the Invention

[0008] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the prior art in the detection of purine substances, such as complex operation, high cost and long time, thereby providing a kit for detecting purine substances quickly, intuitively and stably, as well as its preparation method and application, and a method for detecting or assisting in the detection of purine substances in a sample to be tested using the kit.

[0009] A kit for detecting or assisting in the detection of purine substances comprises reagent 1, reagent 2 and reagent 3, wherein reagent 1 is a color developer, reagent 2 is a reaction enzyme, and reagent 3 is a weak acid salt; the reaction enzyme comprises peroxidase and xanthine oxidase.

[0010] Optionally, the kit further comprises vitamin C and ascorbic acid enzyme; the vitamin C and ascorbic acid enzyme are packaged separately.

[0011] Optionally, the developer consists of a developer A and a developer B, the developer A is 4-AAP, and the developer B is MADB or TBHBA; optionally, the developer A and the developer B are packaged separately.

[0012] Optionally, the reaction enzyme is packaged independently; and the weak acid salt is dipotassium hydrogen phosphate or potassium dihydrogen phosphate.

[0013] The ratio of peroxidase: xanthine oxidase: 4-AAP: MADB or TBHBA is 20-600: 0.1-15: 0.1-5: 0.1-10, and the ratio is KU: KU: g: g.

[0014] Optionally, the ratio of peroxidase: xanthine oxidase: 4-AAP: MADB or TBHBA: vitamin C: ascorbic acid enzyme is 20-1300: 0.1-18: 0.1-5: 0.1-10: 1-12: 1-360, and the ratio is: KU: KU: g: g: mM: KU;

[0015] Alternatively, the ratio of potassium dihydrogen phosphate: peroxidase: xanthine oxidase: 4-AAP: MADB or TBHBA: vitamin C: ascorbic acid enzyme is 0.2-35: 20-1300: 0.1-18: 0.1-5: 0.1-10: 1-12: 1-360, and the ratio is: g: KU: KU: g: g: mM: KU.

[0016] Optionally, the kit further comprises one, two or three of a stabilizer, a surfactant and a preservative.

[0017] Optionally, the stabilizer is selected from one or more of BSA, alcohols or sugars; optionally, the alcohol is any one or more of glycerol, mannitol, ethylene glycol, polyethylene glycol 6000, and sorbitol; optionally, the sugar is any one or more of trehalose, glucose, and sucrose; the surfactant is selected from any one or more of Triton X-100, Twwen-20, or alkyl glycosides; the preservative is NaN3 or Proclin300.

[0018] Optionally, the kit further comprises a uricase inhibitor; optionally, the uricase inhibitor is potassium oxonate;

[0019] Optionally, the kit further comprises bilirubin oxidase;

[0020] Optionally, the kit further comprises independently packaged uricase.

[0021] Optionally, the ratio of peroxidase: xanthine oxidase: 4-AAP: MADB or TBHBA: vitamin C: ascorbic acid enzyme: potassium oxonate is 20-1300: 0.1-18: 0.1-5: 0.1-10: 1-12: 1-360: 0.5-5, and the ratio is: KU: KU: g: g: mM: KU: g;

[0022] Potassium hydrogen phosphate: peroxidase: xanthine oxidase: 4-AAP: MADB or TBHBA: vitamin C: ascorbic acid enzyme: potassium oxonate is 0.2-45: 20-1300: 0.1-18: 0.1-5: 0.1-10: 1-12: 1-360: 0.5-5, and the ratio is: g: KU: KU: g: g: mM: KU: g;

[0023] Optionally, the ratio of potassium dihydrogen phosphate: peroxidase: xanthine oxidase: 4-AAP: MADB or TBHBA: vitamin C: ascorbic acid enzyme: potassium oxonate: bilirubin oxidase is 0.2-35: 20-600: 0.1-18: 0.1-5: 0.1-10: 1-12: 1-360: 1-4: 1-108, and the ratio is: g: KU: KU: g: g: mM: KU: g: KU;

[0024] Optionally, the ratio of potassium dihydrogen phosphate: peroxidase: xanthine oxidase: 4-AAP: MADB or TBHBA: urate oxidase is 0.2-45: 20-1300: 0.1-18: 0.1-5: 0.1-10: 30, and the ratio is: g: KU: KU: g: g: KU.

[0025] Optionally, the box further comprises auxiliary materials, and the auxiliary materials are selected from one or more of lactose, citric acid, sodium bicarbonate, sodium carboxymethyl starch, PVP-K30, PEG6000 and micro-powder silica gel.

[0026] A kit for detecting or assisting in the detection of purine substances, comprising a test paper or a cotton swab; the test paper or the cotton swab contains the reagent of any one of claims 1 to 7.

[0027] Optionally, the kit further comprises a standard colorimetric card and / or a xanthine standard.

[0028] A method for preparing a kit for detecting or assisting in the detection of purine substances, comprising the steps of weighing each component of the kit, dissolving it in water and then fixing the volume, preparing a solution, and packaging it;

[0029] Alternatively, the method may comprise the steps of weighing the various components according to the kit to prepare the target dosage form.

[0030] A system for detecting or assisting in the detection of purine substances, the system comprising the kit or the kit prepared by the method.

[0031] Optionally, the system further includes a biochemical analyzer, an enzyme-labeled instrument, or an ultraviolet-visible spectrophotometer.

[0032] A kit, a kit prepared by the method, and a system for detecting or assisting in the detection of purine substances in a sample to be tested.

[0033] A method for detecting or assisting in the detection of purine substances in a sample to be tested, comprising the step of detecting the sample to be tested using a kit or a system prepared by a kit or method; the method is a non-disease diagnosis method.

[0034] A method for detecting or assisting in the detection of purine substances in a sample to be tested, comprising the following steps:

[0035] 1) Mixing the sample to be tested with the reagents in the kit to form a mixed solution;

[0036] 2) Observe the color of the mixed solution to determine the hypoxanthine / xanthine content in the sample to be tested.

[0037] When the reagent is an effervescent tablet, the sample volume required for testing 1g of effervescent tablet is greater than or equal to 10mL;

[0038] When the reagent in step 1) is a liquid, the required volume of the sample to be tested is 0.3-3 mL;

[0039] Optionally, the method further comprises the steps of adding ascorbic acid enzyme and Vc in sequence.

[0040] Or, optionally, the method further comprises the step of adding potassium oxonate.

[0041] The sample to be tested is food, blood, urine, sweat, saliva, tears or tissue; the blood, urine, sweat, saliva, tears or tissue comes from humans or animals.

[0042] The sample to be tested is blood, urine, sweat, saliva, tears or tissue; the blood, urine, sweat, saliva, tears or tissue comes from an experimental animal; and a urate oxidase inhibitor is also added; the optional urate oxidase inhibitor is potassium oxonate.

[0043] When the sample to be tested is food, the method further includes the step of pre-treating the sample to be tested with a purification reagent.

[0044] A method for detecting or assisting in the detection of purine substances in a sample to be tested, comprising the following steps:

[0045] The sample to be tested is brought into contact with the test paper or cotton swab in the kit, and the concentration of xanthine and hypoxanthine in the sample to be tested is determined based on the color change of the test paper or cotton swab.

[0046] The sample volume required for the test paper method is 0.5-1mL;

[0047] On the test paper before contact, the concentration ratio of potassium dihydrogen phosphate: peroxidase: xanthine oxidase: 4-AAP: MADB or TBHBA is: 1-20: 5-100: 0.01-5: 0.1-5: 0.1-10, and the concentration ratio is: g / L: KU / L: KU / L: g / L: g / L.

[0048] On the test paper before contact, the concentration ratio of potassium dihydrogen phosphate: peroxidase: xanthine oxidase: 4-AAP: MADB or TBHBA: urate oxidase is: 1-20: 5-100: 0.01-5: 0.1-5: 0.1-10: 30, and the concentration ratio is: g / L: KU / L: KU / L: g / L: g / L: KU / L.

[0049] On the cotton swab before contact, the concentration ratio of potassium dihydrogen phosphate: peroxidase: xanthine oxidase: 4-AAP: MADB or TBHBA: ascorbic acid enzyme is: 10-30: 20-400: 0.1-15: 0.1-5: 0.1-10: 1-10, and the concentration ratio is: g / L: KU / L: KU / L: g / L: g / L: KU / L.

[0050] The sample volume required for the cotton swab method is 0.5-1 mL.

[0051] A method for detecting or assisting in the detection of purine substances in a sample to be tested, comprising the following steps:

[0052] (b1) Draw a standard curve

[0053] A series of purine standard solutions are prepared, and several portions of the obtained standard solutions containing different concentrations are mixed with the reagents in the kit to form a mixed solution; the mixed solution is then incubated at room temperature for 30 minutes, and the absorbance of the mixed solution is measured using a microplate reader or a UV-visible spectrophotometer; a standard curve is plotted with the concentration of the purine standard solution as the abscissa and the absorbance of the mixed solution as the ordinate to obtain the standard curve equation;

[0054] (b2) Testing of samples to be tested

[0055] The test sample is mixed with the reagents in the kit to obtain a mixed solution; the absorbance of the mixed solution is measured using a microplate reader, a biochemical analyzer, or a UV-visible spectrophotometer, and the absorbance value of the test sample is substituted into the standard curve equation obtained in step b1) to calculate the concentration of purine substances (xanthine and hypoxanthine) in the test sample.

[0056] When analyzing with a biochemical analyzer, the required sample volume is 0.06-0.2 mL;

[0057] When analyzed by microplate reader, the required sample volume is 0.005 mL;

[0058] When analyzing by UV spectrophotometer, the required sample volume is 0.1 mL.

[0059] The reagents in the kit are prepared as a single dose, a double dose, a triple dose or a quadruple dose;

[0060] When the reagent in the kit is solid, the dosage form can be tablets, capsules or effervescent tablets;

[0061] A single dose consists of all ingredients mixed together.

[0062] The enzyme, stabilizer, color developer and vitamin C can also be mixed together to prepare a freeze-dried preparation to ensure the activity of the enzyme;

[0063] The reagents in the kit are all solid reagents, specifically whole powder, ordinary tablets / capsules / granules or effervescent tablets.

[0064] The pretreatment steps of the purification agent activated carbon (model JH303, powder) are as follows: soaking in 10% NaOH for 3 hours, filtering out, baking at 105° C. for 24 hours for activation treatment, and setting aside.

[0065] The technical solution of the present invention has the following advantages:

[0066] 1. The present invention provides a kit for detecting or assisting in the detection of purine substances, comprising reagent 1, reagent 2 and reagent 3, wherein reagent 1 is a color developer, reagent 2 is a reaction enzyme, and reagent 3 is a weak acid salt; the reaction enzymes include peroxidase and xanthine oxidase. The kit also includes vitamin C and ascorbic acid enzyme; the vitamin C and ascorbic acid enzyme are packaged separately. The kit adds a certain amount of vitamin C to the reagent for detecting purine substances to eliminate the background in urine (a certain amount of xanthine exists in the normal human body, so that normal people are colorless), thereby increasing the degree of differentiation between urine with excessive purine substances (xanthine / hypoxanthine) and normal human urine during detection;

[0067] 2. The present invention provides a method for detecting or assisting in the detection of purine substances in a sample to be tested, comprising the step of detecting a sample to be tested using a kit or a kit or system prepared by the method; the method is a non-disease diagnosis method. When the method detects a sample to be tested, it is fast (short reaction time, only 3 minutes), convenient (easy to carry, suitable for mobile detection), and intuitive (distinguished by the naked eye); the method is used to detect purine substances (xanthine and hypoxanthine) without any instruments and professionally trained personnel to obtain qualitative or semi-quantitative results; when the sample volume is large, the operation is not limited, easy to operate, and the user experience is improved (such as urine samples, which can be directly measured to reduce contact and sampling).

[0068] 3. The reagents in the kit of the present invention can be divided into two, three or four doses, which improves the stability and accuracy of the product; the enzyme can also be made into a lyophilized preparation for immediate use;

[0069] 4. This invention is the first to use purine detection for auxiliary screening and diagnosis of related diseases; it can also play a certain monitoring role in the patient's medication process;

[0070] 5. The present invention provides a system for detecting or assisting in the detection of purines, comprising the above-described kit or the kit prepared by the above-described method; the system also comprises a biochemical analyzer, a microplate reader, or a UV-visible spectrophotometer. There are no similar products on the market that can be used for high-throughput detection.

[0071] 6. The kits, systems and methods provided by the present invention have a wide range of detection, such as relevant biological samples / tissues: blood, urine, sweat, saliva, tears, organs, and other body fluids;

[0072] 7. In animal research, for example, when dissecting animals to detect and evaluate organ function, such as xanthine crystals in the liver and kidneys, xanthine content in joint effusions, and xanthine content in cerebrospinal fluid, the kit, system, and method of the present invention can be used for more intuitive detection with high accuracy.

[0073] 8. The kit, system and method of the present invention can be applied to the detection of foods containing purine, such as fish, beer, vegetables, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0075] Figure 1 (a) shows the results of the experimental group in Example 1 of the present invention; (b) shows the results of the control group in Example 1 of the present invention;

[0076] Figure 2 It is the test result in Example 2;

[0077] Figure 3 It is the test result in Example 3;

[0078] Figure 4 is the standard colorimetric card for the cotton swab method in Example 4;

[0079] Figure 5 is the result of the cotton swab method in Example 4;

[0080] Figure 6 It is the standard colorimetric card for the test paper method in Example 5;

[0081] Figure 7 is the result of the test paper method in Example 5;

[0082] Figure 8 is the standard curve in Example 6;

[0083] Figure 9 is the standard curve of the microplate reader method in Example 7;

[0084] Figure 10 is the standard curve of the UV-visible spectrophotometer method in Example 8;

[0085] Figure 11 is the result of the total xanthine and hypoxanthine content test strip method of the kidney sample in Example 9;

[0086] Figure 12 It is the result of the total xanthine and hypoxanthine content in urine sample;

[0087] Figure 13 This is the result of the stability comparison test of the kit (quantification by microplate reader);

[0088] Figure 14The following are (a) xanthine standard solution, single standard; (b) hypoxanthine standard solution, single standard; (c) adenine standard solution, single standard; (d) guanine standard solution, single standard. DETAILED DESCRIPTION

[0089] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0090] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0091] Reagent specifications and manufacturers are shown in the table below.

[0092] Table 0

[0093]

[0094] Example 1 Visual inspection

[0095] Table 1 Reagent formula

[0096] Reagent 1-1 Reagents 1-2 Reagents 1-3 25g / L dipotassium hydrogen phosphate 15g / L dipotassium hydrogen phosphate 15g / L dipotassium hydrogen phosphate 15mL / L glycerol 8g / L MADB 400KU / L POD 20g / L polyethylene glycol 6000 10KU / L ascorbic acid enzyme 2g / L 4-AAP 3mL / L ethylene glycol 10KU / L XOD 25g / L mannitol 10mM / L Vitamin C 5g / L trehalose 4g / LBSA 5g / L alkyl glycoside (APG) <![CDATA[2 g / L preservative NaN3]]> 4g / L potassium oxonate (OA)

[0097] 1. Preparation: Weigh the corresponding substances according to Recipe 1-1 in the table above and dilute to 1 L with purified water to obtain Reagent 1-1. Similarly, obtain Reagents 1-2 and 1-3. Lyophilize Reagents 1-2 and 1-3 separately into powders and set aside.

[0098] 2. Sample: Use normal urine as a blank control. Add four different concentrations of xanthine standard solutions to the urine to achieve final concentrations of 200 μM, 400 μM, 700 μM, and 1000 μM, respectively.

[0099] 3. Operation: Before use, add 1L of reagent 1-1 to the powdered reagent 1-2 and dissolve and mix thoroughly, which is recorded as reagent R1-A; add 1L of reagent 1-1 to the powdered reagent 1-3 and dissolve and mix thoroughly, which is recorded as reagent R1-B and set aside.

[0100] Take urine and mix it with the above reagent R1-A at a volume ratio of 20:1. After standing for 3 minutes, mix the mixture with reagent R1-B at a volume ratio of 5:1, shake well, and observe the color change after standing for 3-5 minutes (observe the color within 5-30 minutes, as exceeding the limit will affect the result judgment).

[0101] 4. Observation of results: Normal urine and xanthine concentration below 200μM are colorless, while urine with xanthine concentration above 200μM is blue.

[0102] As shown in Figure 1(a), from left to right, the blank control urine before color development, and the urine containing xanthine with final concentrations of 200 μM, 400 μM, 700 μM, and 1000 μM after color development.

[0103] Control group: Vitamin C was removed from the above steps, and other steps remained unchanged. The results are shown in Figure 1(b).

[0104] As can be seen from the above figure, adding vitamin C can eliminate the background in urine (the xanthine content in normal human body is below 160μM). After adding VC, normal human urine appears colorless, which increases the distinction between urine with excessive xanthine / hypoxanthine and normal human urine during detection. Without adding VC, after the urine is colored, the tester needs to subjectively judge whether the xanthine content in the body exceeds the standard based on the depth of the color, which is very easy to cause misjudgment due to interference from light, operation, the color of the urine itself, etc.

[0105] Example 2. Effervescent tablet method detection

[0106] Table 2 Recipe

[0107]

[0108]

[0109] Note: The above percentages are by mass; each kilogram of R2-A contains 10 KU of ascorbic acid enzyme, and each kilogram of R2-B contains 500 KU of POD and 18 KU of XOD.

[0110] 1. Preparation: Mix and evenly press into tablets according to the formula in the above table, 1g per tablet, to obtain effervescent tablets R2-A and R2-B;

[0111] 2. Sample: Using normal urine as a blank control, take 5 aliquots of normal urine and add xanthine standard solution to each aliquot to achieve the final xanthine concentrations of 150 μM, 300 μM, 500 μM, 700 μM, and 900 μM, respectively.

[0112] 3. Operation: Take 150mL of urine sample from step 2 (the sample volume requirement is not high in actual use. If it exceeds 10mL, the effervescent tablet can be dissolved. Alternatively, 2-3 tablets can be directly placed in the toilet to observe the color). Add 1 effervescent tablet R2-A and let it sit for 2 minutes. Then add effervescent tablet R2-B and let it sit for 5 minutes. Observe the color within 30 minutes. The results are shown in the table below. Figure 2, from left to right, the xanthine concentrations are 150μM, 300μM, 500μM, 700μM, and 900μM urine, Figure 2 It can be seen that the xanthine concentration of 150μM does not show color, and the urine with xanthine higher than 150μM turns red. The higher the xanthine concentration in the urine, the darker the color.

[0113] Example 3. Visual inspection

[0114] Table 3 Reagent formula

[0115] Element concentration Dipotassium hydrogen phosphate 30g / L POD 600KU / L 4-AAP 5g / L XOD 15KU / L MADB 10g / L

[0116] 1. Preparation: Weigh the corresponding substances according to the above formula, dilute to 1L with pure water, and obtain formula reagent R3-1, which is ready for use.

[0117] Purification agent activated carbon (model JH303, powdered) pretreatment: soaked in 10g / ml NaOH for 3h, filtered out, and baked at 105℃ for 24h for activation treatment and set aside.

[0118] 2. Preparation of salmon sample solution: Fresh salmon was minced into a paste and divided into 5g portions. One portion was marked as 0h, and the remaining samples were subjected to accelerated decay at 45°C and 75% relative humidity. Take the sample marked as 0h, add 10mL of normal saline, mix thoroughly with magnetic stirring for 5 minutes, and then centrifuge at 8000rpm for 10 minutes. Take the supernatant after centrifugation and add 3mg of activated carbon treated in step 1. Mix thoroughly and let stand for 3 minutes. Remove the supernatant to obtain the 0h salmon sample solution. Then, take a sample at 4h, 8h, 12h, and 18h and process it in the same manner to obtain the 4h, 8h, 12h, and 18h salmon sample solutions.

[0119] 3. Operation: After treatment at different time points, the samples were mixed with the above reagent R3-1 at a volume ratio of 10:1, and placed at room temperature for 10 minutes. The color of the mixture was observed. The results are shown in the table below. Figure 3 .

[0120] 4. HPLC test data comparison:

[0121] Detection conditions: Agilent 1260, C18 column (4.6×250 mm, 5 μm), mobile phase: 10 mM ammonium acetate aqueous solution: methanol = volume ratio 98:2, run time 20 min, flow rate: 1 mL / min, detection wavelength: 270 nm

[0122] Accurate quantitative analysis showed that the purine contents in the salmon sample solutions at 0h, 4h, 8h, 12h and 18h were 26.9μM, 89.7μM, 221μM, 436μM and 605μM, respectively.

[0123] 5. Observation of color development results: The results in step 3 are: as time goes by, the purine content increases and the blue color deepens (from left to right are salmon sample solutions at 0h, 4h, 8h, 12h and 18h).

[0124] In the embodiment, there is no obvious difference in the appearance of the fish meat after being left for 4 hours, but after the detection reagent is added, the blue color of the solution is significantly deepened, which can be used to judge that its purine content is increased; and as time goes on, the blue color continues to deepen. From the liquid phase data, it can be seen that the purine content is positively correlated with the depth of its color, which can be used for semi-quantitative estimation of the purine content in food.

[0125] This shows that the method of this embodiment can perform qualitative / semi-quantitative detection of purine content in food.

[0126] Example 4. Cotton swab method

[0127] Table 4 Reagent formula

[0128] Dipotassium hydrogen phosphate 15g / L MADB 2g / L POD 80KU / L 4-AAP 0.5g / L OA 2g / L XOD 5KU / L Ascorbic acid enzyme 2KU / L Glycerol 4mL / L Polyethylene glycol 6000 10g / L Ethylene glycol 15mL / L Mannitol 5g / L Trehalose 50g / L BSA 2g / L

[0129] 1. Preparation of cotton swabs

[0130] 1. Preparation: Weigh the corresponding substances according to the above formula, dilute to 1L with pure water, and obtain formula reagent R4-1, which is set aside.

[0131] 2. Prepare the cotton swab by the following method: First, fully immerse the blank cotton swab head in R4-1 solution, remove it, and vacuum dry it at 35-40°C for later use. The cotton swab head can be cotton, sponge, or non-woven fabric. In this embodiment, non-woven fabric is used.

[0132] 3. Preparation of Standard Colorimetric Cards

[0133] 1) Prepare xanthine standard solutions at concentrations of 100, 200, 300, 400, 600, and 800 μmol / L;

[0134] 2) Dip the cotton swab prepared in step 2 into each of the six standard solutions for 1 second and immediately remove the swab;

[0135] 3) Prepare a color comparison card based on the color development of each cotton swab after 5 minutes, see Figure 4 .

[0136] 2. Cotton swab method for testing samples

[0137] 1) Samples: Urine samples from healthy individuals were designated as Co1 and Co2; urine samples from gout patients taking 200 mg / d of allopurinol were designated as A1, and urine samples from patients taking 600 mg / d of allopurinol were designated as A2 and A3; urine samples from gout patients taking 40 mg / d of febuxostat were designated as F1, and urine samples from patients taking 80 mg / d of febuxostat were designated as F2 and F3;

[0138] 2) Operation method

[0139] When using, dip the cotton swab into the liquid to be tested for 1 second and immediately remove it (or add the liquid to be tested to the cotton swab), wait for 5 minutes, and compare it with the standard color card ( Figure 4 ) comparison, the concentration corresponding to the standard colorimetric card closest to the test sample color is the concentration of the test solution. If it is between the two color levels, the concentration is estimated.

[0140] 3. HPLC detection of test samples

[0141] HPLC analysis of the sample in step 2: Agilent 1260, C18 column (4.6×250 mm, 5 μm); mobile phase consisting of mobile phase A and mobile phase B, mobile phase A was 10 mM ammonium acetate, mobile phase B was methanol, the volume ratio of mobile phase A to mobile phase B was 98:2, run time was 20 min, flow rate was 1 mL / min, and detection wavelength was 270 nm; the test results are shown in the table below.

[0142] Table 5

[0143] sample Color rendering Estimated concentration using colorimetric cards HPLC test data Healthy People Co-1 Figure 5 (a) <100 μmol / L 37.2 μmol / L Healthy People Co-2 Figure 5 (b) <100 μmol / L 27.5 μmol / L Allopurinol A-1 Figure 5 (c) 200-300 μmol / L 278.2 μmol / L Allopurinol A-2 Figure 5 (d) 400-600 μmol / L 598.4 μmol / L Allopurinol A-3 Figure 5 (e) About 800 μmol / L 759.2 μmol / L Febuxostat F-1 Figure 5 (f) About 200 μmol / L 197.1 μmol / L Febuxostat F-2 Figure 5 (g) About 600 μmol / L 585.2 μmol / L Febuxostat F-3 Figure 5 (h) 600-800 μmol / L 683.9 μmol / L

[0144] From the above table and Figure 5 It can be seen that the results of the cotton swab method in this embodiment are credible.

[0145] Example 5. Test paper method

[0146] Table 6

[0147] Element concentration Dipotassium hydrogen phosphate 5g / L POD 20KU / L 4-AAP 1g / L XOD 0.1KU / L MADB 5g / L Urate oxidase 30KU / L BSA 0.5g / L Polyethylene glycol 6000 1g / L sucrose 3g / L

[0148] 1. Preparation of test paper

[0149] 1) Preparation: Weigh the corresponding substances according to the above formula, dilute to 1 L with pure water, and obtain formula reagent R5-1, which is set aside.

[0150] 2) Test paper preparation

[0151] Soak the test paper substrate strips (Jiulu Polymer Materials Co., Ltd., biological detection test paper) in the above mixed solution R5-1 and shake for 30-60 minutes to allow all components to be adsorbed onto the substrate. Take out the substrate and vacuum dry it at 35-40°C, cut it into test strips with a width of 1 cm and a length of 5 cm, and store it in a dry, sealed, and light-proof plastic bag at low temperature.

[0152] 3) Preparation method of standard colorimetric card

[0153] 1) Prepare xanthine standard solutions at concentrations of 100, 200, 300, 400, 600, and 800 μmol / L;

[0154] 2) Dip the test paper prepared in step 2 into the above standard solution for 2 seconds and then remove it immediately;

[0155] 3) Prepare a colorimetric card based on the color development of each test paper within 5-15 minutes, see Figure 6 .

[0156] 2. Test paper method to detect the sample

[0157] 1) Samples: Urine samples Co1-2 from healthy individuals, urine samples H1-6 from patients with hyperpurinuria

[0158] 2) Operation method

[0159] When using, immerse the test paper prepared in the above steps into the urine sample for 2 seconds and immediately remove it (or drop the urine to be tested onto the test paper), wait for 5 minutes, and compare it with the standard color card ( Figure 6 ) is compared with the color of the standard colorimetric card that is closest to the color displayed by the urine sample to be tested. The concentration corresponding to the color of the standard colorimetric card is the concentration of the urine sample to be tested. If the color is between the two levels, the concentration is estimated.

[0160] 3. Comparison of HPLC test data

[0161] The sample to be tested in step 2 is detected by HPLC.

[0162] HPLC detection was performed using an Agilent 1260 column (4.6 × 250 mm, 5 μm) with a C18 column (4.6 × 250 mm, 5 μm) and a mobile phase consisting of 10 mM ammonium acetate and methanol in a 98:2 volume ratio. The run time was 20 min, the flow rate was 1 mL / min, and the detection wavelength was 270 nm.

[0163] The results are shown in Table 7 and Figure 7 .

[0164] Table 7

[0165] sample picture Estimated concentration using colorimetric cards HPLC test data Healthy People Co-1 Figure 7 (a) <100 μmol / L 28.7 μmol / L Healthy People Co-2 Figure 7 (b) <100 μmol / L 35.3 μmol / L Hyperpurinuria-1 Figure 7 (c) About 400 μmol / L 362.2 μmol / L Hyperpurinuria-2 Figure 7 (d) 600-800 μmol / L 685.7 μmol / L Hyperpurinuria-3 Figure 7 (e) 600-800 μmol / L 655.9 μmol / L Hyperpurinuria-4 Figure 7 (f) About 300 μmol / L 275.2 μmol / L Hyperpurinuria-5 Figure 7 (g) About 300 μmol / L 298.4 μmol / L Hyperpurinuria-6 Figure 7 (h) About 800 μmol / L 783.5 μmol / L

[0166] It can be seen from the above results that the test paper of this embodiment can more accurately detect the total content of purine and hypoxanthine in the urine to be tested.

[0167] Example 6. Kit for biochemical analyzer

[0168] Table 8

[0169] Reagent 6-1 Reagent 6-2 8g / L dipotassium hydrogen phosphate 8g / L dipotassium hydrogen phosphate 0.35g / L MADB 160KU / L POD 10KU / L ascorbic acid enzyme 0.8 g / L 4-AAP 3KU / L bilirubin oxidase 1g / L OA / 0.5KU / L XOD 2mL / L glycerol 2mL / L glycerol 5g / L polyethylene glycol 6000 5g / L polyethylene glycol 6000 10mL / L ethylene glycol 10mL / L ethylene glycol 15g / L mannitol 15g / L mannitol 15g / L trehalose 15g / L trehalose 1g / L BSA 1g / L BSA 1g / L alkyl glycoside (APG) 1g / L alkyl glycoside (APG) <![CDATA[0.5 g / L preservative NaN3]]> <![CDATA[0.5 g / L preservative NaN3]]>

[0170] 1. Preparation of the kit

[0171] 1) Preparation method: Weigh the corresponding substances according to the above formula and dilute to the corresponding concentration with pure water to obtain the formula reagents R6-1 and R6-2 respectively;

[0172] 2) Sample preparation: Urine samples were centrifuged at 12000 rpm for 5 min and the supernatant was collected for testing; serum / plasma samples were centrifuged at 12000 rpm for 5 min and the supernatant was collected for testing; tissue (10 mg) or cells (1 x 10 6 ), added 100 μL of pre-cooled detection buffer (physiological saline), homogenized on ice for 10 min, centrifuged at 12000 rpm for 5 min, and collected the supernatant for detection.

[0173] 3) Preparation of standard curve

[0174] Accurately weigh 4.6 mg of xanthine standard into a 50 mL volumetric flask, ultrasonically dissolve and dilute to volume with pure water to obtain a 604.8 μmol / L xanthine stock solution. The stock solution was diluted 1, 2, 4, 8, 16, 32, and 64 times in sequence to prepare X1-X6 water standards with a concentration range of 9.45-604.8 μM.

[0175] 2. Biochemical analyzer testing

[0176] Biochemical analyzer parameter settings

[0177] Table 9

[0178] Sample / reagent volume (μL) Dilution volume sample 5.6 0 R6-1 48 72 R6-2 20 10

[0179] Using X1-X6 as detection points, a multi-point standard curve was prepared in a Beckman AU480 biochemical analyzer with emission and excitation wavelengths of 660 nm and 800 nm, respectively. Figure 8 .

[0180] 3. Determination of single-standard solutions of xanthine, hypoxanthine, adenine and guanine standards

[0181] Prepare single-label solutions of xanthine standards with final concentrations of 50, 150, 300, and 600 μmol / L;

[0182] Prepare single-label hypoxanthine standard solutions with final concentrations of 50, 150, 300, and 600 μmol / L, respectively;

[0183] Prepare single-label adenine standard solutions with final concentrations of 50, 150, 300, and 600 μmol / L, respectively;

[0184] Prepare single-label guanine standard solutions with final concentrations of 50, 150, 300, and 600 μmol / L, respectively;

[0185] Pipette 50μL of pure water or single standard solution of each standard respectively, add 500μL of R6-1 reagent, mix well and let it stand for 3 minutes, then add 200μL of R6-2 reagent, mix well and let it stand for 10 minutes, and observe the color. Figure 14 , Figure 14 (a) From left to right: 0, 50, 150, 300, 600 μmol / L; Figure 14 (b) From left to right: 0, 50, 150, 300, 600 μmol / L; Figure 14 (c) From left to right: 0, 50, 150, 300, 600 μmol / L; Figure 14 (d) From left to right: 0, 50, 150, 300, 600 μmol / L.

[0186] Example 7. Kit for microplate reader

[0187] Kit composition (100T): 25 mL of detection buffer R7-1; 1 bottle each of reagents R7-2, R7-3, and R7-4; 1 bottle of xanthine standard (1 μM).

[0188] Table 10

[0189] Reagent 7-1 Reagent 7-2 (powder) Reagent 7-3 (powder) Reagent 7-4 (powder) 10g / L dipotassium hydrogen phosphate 0.0003KU XOD 0.0008g TBHBA 0.0007g 4-AAP 1.5g / L OA 0.065KU POD 8mL / L glycerol 0.018KU ascorbic acid enzyme 15g / L polyethylene glycol 6000 0.0054KU bilirubin oxidase 5g / L sorbitol 15g / L glucose 3g / L BSA 2g / L Twwen-20 0.02g / LProclin 300

[0190] Preparation method: Weigh the corresponding substances according to the above formula, dilute to the corresponding concentration with 25 mL of pure water to obtain reagent R7-1; use 220 μL of reagent R7-1 to dissolve 7-2, 7-3 and 7-4 to prepare reagents R7-2, R7-3 and R7-4 respectively (prepare and use immediately)

[0191] Sample preparation: Urine samples were centrifuged at 12000 rpm for 5 min and used directly; serum / plasma samples were centrifuged at 12000 rpm for 5 min and used as standby; tissue (10 mg) or cells (1 x 10 6 100 μL of pre-chilled assay buffer (reagent 7-1) was added to each well and homogenized on ice for 10 minutes. Centrifuge at 12,000 rpm for 5 minutes and collect the supernatant for later use. 5 μL of sample was added to each well and the volume was filled to 50 μL with buffer.

[0192] Prepare the xanthine standard: Dissolve 1 μM xanthine standard in 500 μL of distilled water to a xanthine concentration of 2.0 mM (2.0 nmol / μL). Add 0, 2, 4, 6, 8, and 10 μL of the 2 mM xanthine standard to a 96-well plate, respectively, to achieve a final xanthine concentration of 0, 4, 8, 12, 16, and 20 nmol / well. Add assay buffer to a final volume of 50 μL per well.

[0193] Detection: Add 50 μL of the reaction solution (reagents must be thoroughly mixed before reaction, including 44 μL of R7-1 reagent, 2 μL of R7-2 reagent, 2 μL of R7-3 reagent, and 2 μL of R7-4 reagent) to the well containing the standard and mix thoroughly. Incubate at room temperature in the dark for 30 minutes. Measure the absorbance using a microplate reader at emission and excitation wavelengths of 520 nm and 550 nm, respectively.

[0194] The obtained standard curve is shown in Figure 9 .

[0195] Example 8. UV-visible spectrophotometer kit

[0196] Kit composition (100T): 200 mL of detection buffer R8-1; 1 bottle each of reagents R8-2, R8-3, and R8-4; 1 bottle of xanthine standard (20 μM).

[0197] Table 11

[0198] Reagent 8-1 Reagent 8-2 (powder) Reagent 8-3 (powder) Reagent 8-4 (powder) 15g / L dipotassium hydrogen phosphate 0.006KU XOD 0.012g MADB 0.012g 4-AAP 3g / L OA 1.3KU POD 4mL / L glycerol 0.36KU ascorbic acid enzyme 5g / L polyethylene glycol 6000 0.108KU bilirubin oxidase 6mL / L ethylene glycol 25g / L mannitol 15g / L trehalose 1g / L BSA 1g / L alkyl glycoside (APG) <![CDATA[0.5 g / L preservative NaN3]]>

[0199] Preparation method: Weigh the corresponding substances according to the above formula, dilute to the corresponding concentration with 200 mL of pure water to obtain reagent R8-1; use 5 mL of reagent R8-1 to dissolve 8-2, 8-3 and 8-4 respectively to prepare reagents R8-2, R8-3 and R8-4.

[0200] 2. Sample preparation: Urine samples were centrifuged at 12000 rpm for 5 min and used directly; serum / plasma samples were centrifuged at 12000 rpm for 5 min and used as standby; tissue (10 mg) or cells (1 x 10 6 ), add 100 μL of pre-chilled detection buffer (reagent 8-1), and homogenize on ice for 10 minutes. Centrifuge at 12,000 rpm for 5 minutes, and collect the supernatant for later use. Add 100 μL of sample and make up to 1 mL with buffer.

[0201] 3. Prepare the xanthine standard: Dissolve 20 μM xanthine standard in 10 mL of distilled water to a concentration of 2.0 mM (2.0 nmol / μl). Add 0, 40, 80, 120, 160, and 200 μL of the 2 mM xanthine standard to a 2.5 mL centrifuge tube, respectively, to achieve a final xanthine concentration of 0, 80, 160, 240, 320, and 400 nmol / μL in each tube. Add assay buffer to make up to 1 mL per tube.

[0202] 4. Preparation of standard curve: Add 1 mL of reaction solution (the reagents in the reaction solution must be fully mixed before the reaction, including: 880 μL of R8-1 reagent, 40 μL of R8-2 reagent, 40 μL of R8-3 reagent, 40 μL of R8-4 reagent) to the well containing the standard, mix thoroughly, and incubate at room temperature in the dark for 30 minutes. Measure the absorbance using a UV-visible spectrophotometer (emission wavelength and excitation wavelength are 660 nm and 800 nm, respectively). The obtained standard curve is shown in the figure below. Figure 10 .

[0203] Example 9. Detection of total xanthine and hypoxanthine content in samples after administration of Febuxostat to animals

[0204] 1. Collection of blood samples and kidney tissue: Several rats were gavaged and administered with febuxostat at a dose of 6 mg / kg / day for 7 days. On the 7th day, the rats were anesthetized 1 hour after gavage administration, and blood was collected from the abdominal aorta. Kidney samples were then dissected and collected.

[0205] 2. Urine sample collection: Several rats were gavaged with febuxostat at a dose of 6 mg / kg / day for 7 days. After gavage on the 7th day, the rats were quickly placed in a rat metabolic cage and urine was collected 0-6 hours and 6-24 hours after administration.

[0206] 3. Sample Preparation: Centrifuge urine / blood samples for later use. For kidney samples, add 100 μL of pre-chilled assay buffer (10 mg) and homogenize on ice for 10 minutes. Centrifuge at 12,000 rpm for 5 minutes and collect the supernatant for later use.

[0207] Detection: The visual inspection results were obtained according to Example 3; the cotton swab method results were obtained according to Example 4; the test paper method results were obtained according to Example 5; the biochemical analyzer data were obtained according to Example 6; the microplate reader data were obtained according to Example 7; and the UV-visible spectrophotometer data were obtained according to Example 8; HPLC detection: Agilent 1260, C18 column (4.6×250 mm, 5 μm); the mobile phase consisted of mobile phase A and mobile phase B, mobile phase A was 10 mM ammonium acetate, mobile phase B was methanol, the volume ratio of mobile phase A to mobile phase B was 98:2, the running time was 20 min, the flow rate was 1 mL / min, and the detection wavelength was 270 nm.

[0208] 4. The results of the total xanthine and hypoxanthine content are compared in the table below; the results of the total xanthine and hypoxanthine content of kidney samples are compared in the table below, and Figure 11 , the comparison of the total xanthine and hypoxanthine content in urine samples is shown in the table below, and Figure 12The results showed that there were no statistically significant differences in the biochemical analyzer, microplate reader, and HPLC data for blood samples (P < 0.05). There were no statistically significant differences in the UV-visible spectrophotometer and HPLC data for kidney samples. The test strip and cotton swab methods for urine samples showed similar trends, with no statistically significant differences between the biochemical analyzer and HPLC data (P < 0.05).

[0209] Table 12

[0210]

[0211] Table 13

[0212]

[0213] Example 10. Kit (enzyme reader quantitative) stability comparison test

[0214] The reagents of Example 7 were evenly divided into 13 groups, and 13 groups of commercial xanthine / hypoxanthine detection kits (Sigma, product code MAK186) were taken as controls; they were placed in a refrigerator at 2-8°C. On the same day of each month, one group of reagents was taken out to test the xanthine standard solution (target value 100 μM / L). The test results showed that the reagents of Example 7 were more stable than the common xanthine / hypoxanthine detection kits on the market under storage conditions of 2-8°C. The results are shown in the table. Figure 13 .

[0215] Comparative Example 1

[0216] Sample processing: Take normal urine and add xanthine standard solution to make the final concentration of xanthine in urine 500μM and 1000μM respectively. Add activated carbon to the urine at an amount of 0.5g / mL, mix well, centrifuge and take the filtrate for HPLC detection. Each concentration was repeated 10 times. The HPLC detection results are shown in the table below:

[0217] Table 4

[0218]

[0219] The above table shows that activated carbon randomly adsorbs xanthine without regularity and cannot achieve quantitative removal. The results are consistent when activated carbon is added at different concentrations.

Claims

1. A kit for detecting or assisting in the detection of purine substances, characterized in that: The method comprises reagent 1, reagent 2 and reagent 3, wherein reagent 1 is a color developer, reagent 2 is a reaction enzyme, and reagent 3 is a weak acid salt; the reaction enzyme comprises peroxidase and xanthine oxidase; The kit further comprises vitamin C and ascorbic acid enzyme; the vitamin C and ascorbic acid enzyme are packaged separately; The developer consists of developer A and developer B, developer A is 4-AAP, and developer B is MADB or TBHBA; Peroxidase: Xanthine oxidase: 4-AAP: MADB or TBHBA: Vitamin C: Ascorbic acid enzyme is 20-1300: 0.1-18: 0.1-5: 0.1-10: 10:10, the ratio is: KU:KU:g:g:mM:KU.

2. The kit for detecting or assisting in the detection of purine substances according to claim 1, characterized in that The developer A and developer B are both packaged independently.

3. The kit for detecting or assisting in the detection of purine substances according to claim 1, characterized in that The reaction enzyme is packaged independently; the weak acid salt is dipotassium hydrogen phosphate or potassium dihydrogen phosphate.

4. The kit for detecting or assisting in the detection of purine substances according to claim 3, characterized in that: In the kit, the ratio of peroxidase: xanthine oxidase: 4-AAP: MADB or TBHBA is 20-600: 0.1-15: 0.1-5: 0.1-10, and the ratio is KU: KU: g: g.

5. The kit for detecting or assisting in the detection of purine substances according to claim 3, characterized in that: Potassium hydrogen phosphate: peroxidase: xanthine oxidase: 4-AAP: MADB or TBHBA: vitamin C: ascorbic acid enzyme is 0.2-35: 20-1300: 0.1-18: 0.1-5: 0.1-10: 10:10, the ratio is: g:KU:KU:g:g:mM:KU.

6. The kit for detecting or assisting in the detection of purine substances according to claim 1, characterized in that: The kit further comprises one, two or three of a stabilizer, a surfactant and a preservative.

7. The kit for detecting or assisting in the detection of purine substances according to claim 6, characterized in that: The stabilizer is selected from one or more of BSA, alcohols or sugars; The alcohol is any one or more of glycerol, mannitol, ethylene glycol, polyethylene glycol 6000, and sorbitol; The sugar is any one or more of trehalose, glucose, and sucrose; the surfactant is any one or more of Triton X-100, Twwen-20, or alkyl glycoside; and the preservative is NaN3 or Proclin 300.

8. The kit for detecting or assisting in the detection of purine substances according to claim 1, characterized in that: The kit further comprises a uricase inhibitor; the uricase inhibitor is potassium oxonic acid.

9. The kit for detecting or assisting in the detection of purine substances according to claim 8, characterized in that: The kit also includes bilirubin oxidase.

10. The kit for detecting or assisting in the detection of purine substances according to claim 8, characterized in that: The kit further comprises urate oxidase in a separate package.

11. The kit for detecting or assisting in the detection of purine substances according to claim 8, characterized in that: Peroxidase: xanthine oxidase: 4-AAP: MADB or TBHBA: vitamin C: ascorbic acid enzyme: potassium oxonate is 20-1300: 0.1-18: 0.1-5: 0.1-10: 10: 10: 0.5-5, and the ratio is: KU: KU: g: g: mM: KU: g; The ratio of potassium dihydrogen phosphate: peroxidase: xanthine oxidase: 4-AAP: MADB or TBHBA: vitamin C: ascorbic acid enzyme: potassium oxonate is 0.2-45:20-1300:0.1-18:0.1-5:0.1-10:10:10:0.5-5, and the ratio is: g:KU:KU:g:g:mM:KU:g.

12. The kit for detecting or assisting in the detection of purine substances according to claim 9, characterized in that: The ratio of potassium dihydrogen phosphate: peroxidase: xanthine oxidase: 4-AAP: MADB or TBHBA: vitamin C: ascorbic acid enzyme: potassium oxonate: bilirubin oxidase is 0.2-35:20-600:0.1-18:0.1-5:0.1-10:10:10:1-4:1-108, and the ratio is: g:KU:KU:g:g:mM:KU:g:KU.

13. The kit for detecting or assisting in the detection of purine substances according to claim 12, characterized in that: The ratio of potassium dihydrogen phosphate: peroxidase: xanthine oxidase: 4-AAP: MADB or TBHBA: urate oxidase is 0.2-45: 20-1300: 0.1-18: 0.1-5: 0.1-10: 30, and the ratio is: g: KU: KU: g: g: KU.

14. The kit for detecting or assisting in the detection of purine substances according to claim 1, characterized in that: The box further comprises auxiliary materials, which are selected from one or more of lactose, citric acid, sodium bicarbonate, sodium carboxymethyl starch, PVP-K30, PEG6000 and micro-powder silica gel.

15. A method for preparing a kit for detecting or assisting in the detection of purine substances according to any one of claims 1 to 14, characterized in that: The method comprises the steps of weighing each component according to the kit according to claims 1 to 14, dissolving the components in water and then fixing the volume to prepare a solution, and packaging the solution.

16. A system for detecting or assisting in the detection of purine substances, the system comprising the kit according to any one of claims 1 to 14 or the kit prepared by the method according to claim 15.

17. The system according to claim 16, wherein: The system further comprises a biochemical analyzer, an enzyme-labeled instrument or an ultraviolet-visible spectrophotometer.

18. A method for detecting or assisting in the detection of purine substances in a sample, characterized in that: The method comprises the steps of detecting a sample to be tested using the kit described in any one of 1-14 or the kit prepared by the method described in claim 15 or the system described in claim 16 or 17; the method is a non-disease diagnosis method.

Images